© 2015 Nature America, Inc. All rights reserved. addressed addressed to H.M. ( 4 Genomics, Duke University Medical Center, Durham, North Carolina, USA. 1 vation odor. given by a activated ORs of number between the correspondence throughput humans in perception odor and mice in tion that shown OR10G4 have humans, and in OR2J3 OR5A1, OR11H7, OR7D4, and mice, in SR1 OR-EG, and I7, MOR23 of M72, M71, those including many studies, on relies ORs lian revealed diverse odor tuning properties among the tested ORs ligands. Notably, molecular receptive range analyses of a few ORs have in vitro RT-PCR cell pression in vivo, ex vivo hensive identification of ORs that respond to a given odorant stimuli. of olfactory number a large discriminate and detect of nation ORs be activated by a set of odorants and one odorant can activate a combi recognition follows a combinatorial coding scheme, where one OR can lium (OE). The mouse over genome encodes ORs intact 1,000 epithe olfactory the in located (OSNs) neurons sensory olfactory in protein–coupled receptors (GPCRs) ands predominantly by ORs, a large family of seven transmembrane G with Instarts sensation of the detection olfactory mammals, odor lig Our results provide a method for the understanding coding combinatorial of odors residues specific to the acetophenone or TMT receptors and developed models to predict receptor activation by acetophenone. (TMT), encompassing 69 OR-odorant pairs. 2,5-dihydro-2,4,5-trimethylthiazoline We also identified shared amino acid In combination with validations in a heterologous system, we identified sets of ORs for two odorants, acetophenone and screened the endogenously expressed ORs against an odor in one set of experiments using awake and freely behaving mice. of ribosome immunoprecipitation mRNA from olfactory epithelium of odor-stimulated mice followed by RNA-Seq. This approach We developed a means of high-throughput volatile odor molecules. odor Understanding coding requires mapping comprehensive between ORs and odors. corresponding The mammalian olfactory system uses a large family of odorant receptors (ORs) to detect and discriminate amongst a myriad of Hiroaki Matsunami Yue Jiang identifies odorant receptors for odors Molecular profiling of activated olfactory neurons nature nature Received 19 June; accepted 9 August; published online 31 August 2015; Department Department of Neurobiology, Duke Institute for Brain Sciences, Duke University Medical Center, Durham, North Carolina, USA. should Correspondence be Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA. Currently, large-scale identification of active ligands for mamma for ligands active of identification large-scale Currently, The S6 ribosomal subunit is phosphorylated following neuronal acti Deciphering the coding of olfactory information requires the compre 1 OR expression 7 . This phosphorylation is comparable to induction of immediate 6 NEUR , 7 , calcium imaging of dissociated OSNs combined with single- 1 in vivo in 3 – , 7 and 3 3 OSCI , the use of fluorescently labeled transgenic mice transgenic labeled fluorescently of use the , , , , Naihua Natalie Gong 4 . Through such combinatorial coding, mammals can mammals coding, such combinatorial . Through [email protected] in vitro mapping methods makes it difficult to estimate estimate to difficult it makes methods mapping EN in vitro in 9 C have been used to match ORs with their cognate E

1 methods, such as virus-mediated OR overex , 4 advance online publication online advance heterologous cell systems cell heterologous in in vitro in vitro in 1 , and 2 responses predict OSN activa OSN predict responses . ORs are individually expressed in in vivo ). 1 , , Xiaoyang Serene Hu 12– in in vivo results across a across results large 1 6 , the lack of high- of lack the , identification of identification OR repertoires responding to odorants using phosphorylated 4 , 9 , 1 2

. Though Though . 5 . Various 10 2 . . Odor , 1 doi:10.1038/nn.410 3 8 1 Department Department of Statistical Science, Duke University, Durham, North Carolina, USA. and . ------

1 , , Mengjue Jessica Ni OE. We presented each of the tested mice with a stimulation cassette cassette a stimulation with mice We OE. tested of the each presented the in phosphorylation S6 to leads stimulation odor whether tested when ORs occurs are in activated the OSNs, phosphorylation we first S6 odor. Towhether tested to the respond ORs determine associated occurs in the OSNsphosphorylation activated by ribosome odor exposure and, if whether so, whether pS6- unknown is the it to However, odor. responding be likely would OSNs activated with associated ORs as ORs, odor-activated map to system olfactory the in applied over of genes OR out possible 1,000 allele OR one express to chooses OSN each OE, the In Odor exposure leads to S6 phosphorylation in the OE RESULTS TMT. and acetophenone to responding and mapping in mRNAs OR of high-throughput both Using odorants. with subsets stimulated mice in enrichment revealed acti pS6-IP OSN for vation. marker reliable a is and stimulation odor following of neurons that respond to feeding, starvation and high salt applied in brain regions such as the hypothalamus to identify markers expressed in activated neurons ing with RNA-Seq has been developed as a method to identify mRNAs phospho-S6 immunoprecipitation (pS6-IP) followed by mRNA profil associated with mRNA species expressed in thephysically being activatedof advantage the neurons.has S6 phosphorylated expression,Thus, neurons active mark to as such expression, gene early We found that S6 phosphorylation occurs in the mouse OE OE mouse the in occurs phosphorylation S6 that found We 4 in vitro in in in vivo validation, we identified diverse sets of ORs ORs of sets diverse identified we validation, 15 in vivo . 3 1 , 18 , 2 , Radhika Pasi , Radhika 0 , 2 1 . Thus, we reasoned that pS6-IP could be be could pS6-IP that reasoned we . Thus, University University Program in Genetics and 9 . However, . gene early immediate unlike 1 c-Fos 7 . This approach has successfully been and Egr-1 1 & , which is widely used used widely is which ,

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© 2015 Nature America, Inc. All rights reserved. indicate colocalization of Olfr160 and pS6 signals. Scale bar represents 25 represents antibody staining for pS6, magenta represents RNA FISH for 5 ORs, and blue represents bisbenzimide staining showing the nuclei. Arrows acetophenone) and controls (no odor, heptanoic acid, TMT, (+)-carvone). ( 25 and blue represents bisbenzimide staining showing the nuclei. Arrowheads indicate colocalization of Olfr160 and pS6 signals. Scale bars represent (control) condition for 1 h. Green represents antibody staining for pS6, magenta represents antibody staining for the known acetophenone receptor Olfr160, Figure 1 (fraction of OSNs showing positive pS6 staining following stimulation pS6 for staining strong displayed OE stimulated the in OSNs of tion subpopula a OE, control the in low was signal pS6 ( background the antibody anti-pS6 with OE the from sections coronal stained and later h 1 animals clean the We a killed cage. in disposable (control) water distilled or (odor), acetophenone 1% or 10 with spotted paper filter of piece a enclosing t r a  pairs. ( d b e a

Olfr1 Corrected total cell Meth Meth µ Olfr690(MOR31 m. ( Corrected total –2 fluorescence A No-odor contr Heptanoic acid(1/70) 60(M72) 0 2 4 cet

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–2 Olfr961(MOR224-5) (+)-C ) Quantification of pS6 staining intensity following 1% and 100% odorant stimulation for five known OR-odorant pairs. Error bars indicate s.d.

ophenone (34/41) C I b 0 2 4 Odor stimulation induces S6 phosphorylation in the mouse OE. ( ) Quantification of pS6 induction in Olfr160-expressing OSNs following odor stimulation by Olfr160 agonists (methyl salicylate, methyl benzoate, No odor Acetophenone 1 h ar oat 0%

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Olfr1 v –2 aler 0 2 4 440(MOR21 Olfr2(l7) ic acid Olfr961 (MOR224–5): 0% 1 00% c a ) Coronal section of OE stimulated with 100% acetophenone for 1 h. Green ) Coronal section of OE stimulated with 100% acetophenone (upper) and no odor

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Olfr690(MOR31 induction pS6 of specificity the determine To stimulation. none acetophe to response in phosphorylation S6 show OSNs expressing Olfr160- that suggesting signals, pS6 and Olfr160 of colocalization tophenone receptor M71 is a pseudogene in C57BL/6 strain), revealed as known also receptor,M72 related ace (the acetophenone Olfr160, 6 16 acetophenone: 100% with –2 d ± 60(M72) 0 2 4 ) Quantification of pS6 staining intensity for five known OR-odorant Olfr961(MOR224-5) 2%, 1 00% n -2) = 3 images). Double staining with an antibody against a known c (MOR224-5) Olfr1 (MOR21 (MOR31 Eugenol advance online publication online advance Olfr1 Olfr2(l7) Olfr961 Olfr690 Olfr1

440(MOR21 (M72) Olfr2(l7) –2 0 2 4 440 60 5-1) -2)

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© 2015 Nature America, Inc. All rights reserved. revealed that pS6 signals started by 30 min and reached a plateau plateau a reached and min 30 by started signals exposure pS6 that acetophenone revealed after signals pS6 of analysis Time-course eugenol ( Dunnett’s and ( odorants, was observed pS6 induction significant cognate of 1%) and (100% concentrations different two ANOVADunnett’s and ( combinations OR-odorant control with pared com induc pairs OR-odorant pS6 reported previously with significant observed was tion probes, OR the of each with labeled OSNs 2a Fig. ( immunostaining pS6 by followed performed and stimulated individual mice with one of the odorants (10 We and Olfr1440). (Olfr2 expressed and were two ventrally Olfr961) and Olfr690 (Olfr160, expressed dorsally were ORs Three odorants. non-activating with ORs of pairs control and acetophenone) (M72, muscone) (MOR215-1, Olfr1440 and acid) isovaleric (MOR31-2, Olfr690 including pairs, OR-odorant known for intensity five staining using pS6 induction we quantified method, ( signals Olfr160 with overlap little showed acid, signals pS6 (heptanoic odors (+)-carvone), TMT, control by stimulated animals in whereas, OSNs, Olfr160-expressing of proportion large a in phosphorylation led methyl acetophenone) to benzoate, agonists S6 (methyl salicylate, that showed pS6 staining. As odor expected, stimulation with Olfr160 1a Fig. other with mice the agonists Olfr160 known stimulated we exposure, odor to response in indicates ( group. acetophenone–stimulated 1% in mRNA OR of ( slope. unit represents line Broken repertoire. OR detected the across comparisons multiple for adjusting with group acetophenone–stimulated 1% the in enriched were ORs 25 mRNA. OR of calling enrichment ( group. acetophenone–stimulated 100% in mRNA OR of enrichment showing plot ( slope. unit represents line Broken repertoire. OR detected the across comparisons multiple for adjusting after 0.05, than smaller with group acetophenone–stimulated 100% the in enriched were ORs 75 mRNA. ( slope. unit the represents line broken the genes non-OR represent dots gray ORs, represent ( samples IP stimulated acetophenone in genes of counts read mean ( samples IP unstimulated x sample. unstimulated versus acetophenone) (100% sample stimulated from counts mRNA ( RNA-Seq. by profiled be then could which OSNs, activated the in expressed species mRNA for enriched pS6-IP OSNs. odor-responding in phosphorylation underwent S6 subunit odor, to ribosome exposed was animal the When ( OE. stimulated 2 Figure nature nature b axis, mean read counts of genes in genes of counts read mean axis, ) Scatter plot comparing immunoprecipitated immunoprecipitated comparing plot ) Scatter To further evaluate the specificity and sensitivity of the pS6 pS6 the of sensitivity and specificity the evaluate further To 2 2 c , Olfr961 (MOR224-5, eugenol) (MOR224-5, Olfr961 , ) and quantified the proportion of Olfr160-expressing OSNs Olfr160-expressing of proportion the quantified and ) g ). When we quantified pS6 signal intensities in individual individual in intensities signal pS6 quantified we When ).

) Differential enrichment calling of OR of calling enrichment ) Differential ) Scatter plot comparing comparing plot ) Scatter Fig. 1 Fig. NEUR pS6-IP enriches OR mRNAs from odor odor from mRNAs OR enriches pS6-IP P Fig. 1 = 0.001 and the blue dashed line represents represents line dashed blue the and = 0.001 P f value smaller than 0.05, after after 0.05, than smaller value ) Volcano plot showing enrichment enrichment showing plot ) Volcano OSCI b e post hoc post a and and in situ in , , ) Scheme of the experiment. experiment. the of ) Scheme Supplementary Supplementary Fig. 3 EN Supplementary Fig. 1b Fig. Supplementary n hybridization with each of the five OR probes probes OR five the of each with hybridization C = 3). Red dots dots Red = 3). test), with the exception of Olfr961 with 1% 1% with Olfr961 of exception the with test), E post hoc post 2 n

1 = 3). = 3). along with control odors ( control odors with along advance online publication online advance y P e axis, axis, test). When we tested the ORs with with ORs the tested we When test). d values of enrichment in 100% acetophenone– versus 1% acetophenone–stimulated samples. The red dashed line line dashed red The samples. acetophenone–stimulated 1% versus acetophenone– 100% in enrichment of values ) Differential ) Differential ) Volcano ) Volcano P values values i. 1c Fig.

and 15

2 P , 2

2 , Olfr2 (I7, heptanal) (I7, Olfr2 , < 0.001, one-way ANOVA one-way < 0.001, 4 , in addition to Olfr160 Olfr160 to addition in , Supplementary Supplementary Table 1 ). , d and and P a e b < 0.0001, one-way one-way 0.0001, < log10(mean read counts, log10(mean read counts, Supplementary Supplementary

Supplementary Supplementary stimulated) 0. 2. P 1. 1. stimulated) 0 1 2 3 4 5 5 0 0 0 5 = 0.05. Note the absence of ORs in the bottom right corner. right bottom the in ORs of absence the Note = 0.05. µ 1 0 0 0 l, undiluted) lo lo

g g 10 .5 10 no-odor cont no-odor cont (mean (mean 1. 2 6 1 0 , 8 re re , P P 2 ad counts ). ). ad counts Odor .5 <0.00 <0.05 3 - - 4 3

ro ro l) 2. l) O 2 0 suggesting that the enrichment process is reproducible and is is and ORs additional vate reproducible is process acti that concentrations odor high findings previous with consistent enrichment the that suggesting with enriched ORs for 9 of (9 stimulation acetophenone 100% following enriched also were ORs these of Most adjusted). FDR respectively, ( none adjusted; (FDR) with ORs 75 10 with stimulation following enriched significantly were that (M72), Olfr160 including stimulated the in ORs, of set large a enriched identified analysis was expression ORs Differential group. of subset a in groups, levels two comparable at the expressed Although were genes of transcripts. OR majority vast of the exons coding to mapped that reads OSNs activated ( in species mRNA enriches pS6-IP that suggesting genes early immediate the 10 with stimulated mice from samples of the RNA. Comparison purified the tissue, the analyzed and RNAs homogenized associated with later, along pS6 h immunoprecipitated 1 OE the dissected We control. odor no or acetophenone either same to exposed experiment. were the mice pair, RNA-Seq each In of pS6-IP each for littermates used of were age, consisting and gender each mice, of pairs Three pS6- the by followed RNA-Seq in pS6-IP OSNs, ( we positive performed expressed ORs the identify comprehensively more To stimulation odor following mRNAs OR enriches pS6-IP stimulation. odor following activation OSN for marker reliable ( h 1 at 1 i. 2 Fig. ( n ( , , n =25) Other Egr1 c-F ORs =9) .5 5 os P s OE < 0.001, 14 of 25 for ORs enriched with with enriched ORs for 25 of 14 0.001, < Fig. 2e Fig. Supplementary Fig. 2b Fig. Supplementary b c µ pS6 ). We measured expression levels for ORs by counting counting by ORs for levels expression measured We ). log (mean read counts,

l of acetophenone versus control revealed a higher level of level higher a revealed control versus acetophenone of l 10 f –log (P value) stimulated)

10 0. 2. 2. 1. 1. 20 25 30 15 10 P 5 0 5 0 5 0 0 5 , < 0.001 and and < 0.001 f mRNA inunactiv mRNA inactiv 0 ) enriched 9 and 25 ORs ( ORs 25 and 9 enriched ) Fig. 2c Fig. P P lo –4 <0.05 <0.00 g 0. 10 3 5 no-odor contr P lo –2 (mean r , µ 2 g at 5 at 2 l of undiluted (100%) acetophenone (47 and and (47 acetophenone (100%) undiluted of l ed OS 1. 1 (f ed OS . ( 0 n old change) ( 6 4 2 0 , n =25) d N =9) ead count N ). Stimulation with 10 10 with Stimulation ). 1. P P c-Fos P 5 <0.05 <0.00 < 0.05, respectively, false discovery rate discovery false respectively, < 0.05, ol 2. ) 0 ). These results suggest that pS6 is a is pS6 that suggest results These ). and and 1 s, ( n ( 2. n =75) =47) 5 g Egr1 log10(P value, 100% stimulation) P d –20 –25 –30 –1 –1 in the stimulated samples, samples, stimulated the in –5 –log (P value) 0 5 0 10 20 25 15 10 P pS6-IP 0 5 0 < 0.001 and and 0.001 < log –6 –5 10 P P ( <0.05 <0.00 µ P –4 P –1 t r a

l of 1% acetophe 1% of l va < 0.05; 0.05; < 0 log lue –2 –1 1 2 ( (f 5 ,1% stimulation) n ( old change) n =75) –20 0 =47) RNA C I 6 4 2 –25 P Fig. 2 Fig. P P -Seq Fig. Fig. 2 < 0.05, 0.05, < =0.05 =0.00 –30 e l –35 1 g a 8 s ), ), ). ).  - -

© 2015 Nature America, Inc. All rights reserved. in vitro in 3 Fig. as a groupanalysis ( pS6-IP the in acetophenone by activated were that ORs the P enriched ORs was higher than that of control ORs ( stimulation. As expected, acetophenone-induced control with compared as activity luciferase of fold-increase relative controls. We quantified the degree of OR activation by determining the cells with 3 expression of various transiently expressed ORs cells, which are an HEK293T-derived cell line that supports the robust enriched ( with ORs 75 (of acetophenone 100% by enriched ORs 71 tested we none, test whether the enriched ORs are more likely to respond to acetophe ( acetophenone to ORs these of response luciferase reporter gene assay in heterologous cells activated ORs, we leveraged the previously established cAMP-mediated To confirm that the acetophenone to respond to tend ORs Enriched no better than random). tailed against H P the OR was enriched at using curves illustrating performance of classifiers performance no better than random). ( rank-sum test (one tailed against H in vitro predict whether the OR responds to acetophenone classifiers using ( activated: of percentiles. Wilcoxon rank-sum test for difference percentiles, and whiskers represent 5th and 95th represent median, boxes represent 25th and 75th indicates ORs not activated IP. Red indicates ORs activated y of 107 activated and 413 not activated ORs. 300 unenriched: 3 of percentiles. Wilcoxon rank-sum test for difference percentiles, and whiskers represent 5th and 95th represent median, boxes represent 25th and 75th indicates ORs not enriched ( Red indicates ORs enriched at empty vector stimulated with 3 0% was determined by the fold of increase of Olfr1126 stimulated with 300 100% was determined by the fold of increase of normalized fold of increase in luciferase signals. 71 enriched and 449 unenriched ORs. acetophenone. ( ( RTP1S, receptor-transporting protein 1 (short). element–binding protein; PKA, protein kinase A; response element; CREB, cAMP response pathway. AC, adenylyl cyclase; CRE, cAMP responses. ( Figure 3 t r a  was which cut-off, the of determination the for Methods Online (see e b axis, fold of enrichment of transcripts by pS6 = 7 × 10

= 7 × 10 × 7 = ) ROC curves illustrating performance of ) ) ORs tested for in vivo in vitro µ P in vitro M d . AUC: 0.754, < 0.05 after FDR correction) and 449 control ORs that were not ). The cut-off for defining positive response to acetophenone acetophenone to response positive defining for cut-off The ).

was a 2.33-fold increase in luciferase induction at 300 300 at induction luciferase in increase 2.33-fold a was C I P enrichment between activated and not responses between enriched and Correlation between P = 7 × 10 −21 Fig. 3 −21 = 10 a µ responses to predict whether ) The heterologous OR signaling e l , Wilcoxon rank-sum test (one M, M, 30 µ at 300 at 300 0 M c in vivo : classifier performance −14 ) b P in in vitro s In vitro ). We independently expressed these ORs in Hana 3A −21 = 0.1, 30 . N.S., µ in vivo P µ . ( M M or 300 enrichment = 6 × 10 P M, Wilcoxon rank-sum test; Wilcoxon test; M, rank-sum d < 0.05. AUC: 0.845, responses to activation of ) P In vivo in vitro > 0.05; *** P receptor mapping identified acetophenone- > 0.05). Black bars in vivo µ P µ µ in vitro M M acetophenone. < 0.05 and gray −16 M acetophenone. µ enrichment P . Black bars 0 M M of acetophenone as well as no-odor P , Wilcoxon : classifier = 0.005, and values to P f and gray ) ROC = 10 y

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9 in vitro in , 2 a 6 in vitro ). To systematically Tosystematically ). e c a , and stimulated the 26 P Fig. 3 Fig. Normalized in vitro activation

= 0.005 at 30 True positive rate , 2 100 120 0.2 0.4 0.6 0.8 1.0 CREB 7 in vitro in 20 40 60 80 ATP to measure the were enriched enriched were 0 0 activation of AC c 0 ). Similarly, ). cAM PKA Not enriched( Enriched ( AUC: 0.754 3 N.S. P µ 0.2 CREB M µ µ Golf M, M False positiverate n Random In vivo =71) - P

0.4 n 30 =449) o CRE OR *** phorylation analysis with acetophenone, expressed these ORs in in ORs these expressed acetophenone, with analysis acetophenone- phorylation the phos S6 the investigate in enriched were that ORs further We selected ORs. to activated out set we between responses, concordance the establishing After ORs acetophenone of Identification against tailed H RNA-Seq(AUC0.845, = vitroIn against H tailed ROC curve (AUC) = 0.754, acetophenone to responded OR the whether predicted RNA-Seq pS6-IP in Enrichment varied. is predictor the of positive rate against the false positive rate as the discrimination cut-off ated receiver operating characteristic (ROC) curves by plotting the true tify how well our of enrichment 13% (58 ORs) responded. 74% (43 ORs) of these 58 ORs showed trends by 100% acetophenone ( and 69% (49 ORs) responded. In contrast, of the 1,047 ORs not 100% acetophenoneenriched experiment ( set to exclude 99% of negative receptors). Of the 75 ORs enriched in the Odor enrichmentclassifier µ M 0.6 Luciferase RTP1S response also predicted the the predicted also response 300 *** 0.8 advance online publication online advance µ M in vivo 0 0 1.0 in vivo : classifier performs no performs better than : random; classifier : classifier performs no better than random; no better performs : classifier , but did not reach statistical significance. To quan and f d b P log (mean read counts, stimulated) > 0.05), 449 ORs were tested True positive rate Fold enrichment in vivo P 10 100 = 7 × 10 × 7 = 0.2 0.4 0.6 0.8 1.0 0.5 1.0 1.5 2.0 2.5 P in vitro 20 40 60 80 0 0 0 = 6 × 10 0 0 log In vitro P AUC: 0.845 10 Not activated Activated results predict each other, we gener < 0.05), 71 ORs were tested (mean readcounts,no-odorcontrol) −21 P 0.5 −16 0.2 *** value of enrichment in pS6-IP pS6-IP in enrichment of value response , Wilcoxon rank-sum test, one test, Wilcoxon rank-sum , , Wilcoxon rank-sum test, one False positiverate in vitro

1.0 Random In vitro 0.4 in vitro nature nature in vitro in ( n responseclassifier n vivo in =107) 1.5 ( FDR adjusted FDR adjusted Olfr160 (M72) Tested 0.6 n =413) (area under the the under (area NEUR in vitro 2.0 in vitro and and 0.8 OSCI and only P P 2.5 Fig. Fig. 3 Fig. Fig. 3 n vitro in <0.001 <0.05 in vitro 1.0 EN f e C ). ). ). E - - -

© 2015 Nature America, Inc. All rights reserved. are likely to be bona fide acetophenone receptors. acetophenone fide bona be to likely are both by supported receptors, 48 these ( receptors acetophenone 48 of total at identified thus experiments stimulation 100% and 1% The enrichment. toward trends showed they although significance, by enriched also were did not statistical reach three other the whereas acetophenone, 100% (80%) 12 acetophenone, 1% for ORs identified acetophenone confirmed 15 the Of responded. (63%) 15 and ( experiment acetophenone 1% in were tested the 75 ORs in enriched 100% acetophenone ( experiment ( acetophenone by activated indeed were ORs confirmed 48 that profiles response luciferase dose-dependent The none. Hana3A cells nature nature acetophenone to respond ventral OE (Olfr736 and Olfr1093), along with a control OR (Olfr1132) following 1% and 100% acetophenone stimulation. acetophenone 100% and 1% following (Olfr1132) OR a control with along Olfr1093), and (Olfr736 OE ventral and Olfr1444) Olfr1104, Olfr923, (Olfr19, OE dorsal the in expressed ORs acetophenone identified newly several expressing OSNs in induction pS6 of 25 represents bar Scale nuclei. the showing staining bisbenzimide indicates blue and Olfr19 OR acetophenone identified newly ( 1. as defined was Olfr160 of response maximum The s.e.m. indicate bars Error (M72). Olfr160 to scaled were Responses receptors. acetophenone the ( samples 4 Figure c c b a ) Coronal section of OE following acetophenone stimulation for 1 h. Green indicates antibody staining for pS6, magenta indicates RNA FISH for the the for FISH RNA indicates magenta pS6, for staining antibody indicates Green 1 h. for stimulation acetophenone following OE of section ) Coronal To confirm that the acetophenone ORs that we identified do do identified we that ORs acetophenone the that confirm To Acetophenone Response Read counts

0.1 0.2 0.3 0.4 0.5 0.6 Response 100% 1% 0% 200 400 0 2 4 6 8 0

Olfr923 0 – –

∞ ∞

log Olfr749 log pS6 NEUR Identification of acetophenone receptors. ( receptors. acetophenone of Identification n Olfr1047 Olfr1104 Olfr874 Olfr935 Olfr1 Olfr205 Olfr1484 Olfr744 Olfr1377 Olfr145 –9 –9

= 3 pairs). Right, read counts of acetophenone receptors in control and 100%–stimulated samples ( samples 100%–stimulated and control in receptors acetophenone of counts read Right, = 3 pairs). Olfr491 10 10 [acetophenone] (M in in vitro [acetophenone] (M) Olfr136 9 –8 –8

9 Olfr1377 + Bisbenzimide and challenged the and with cells 10 challenged OSCI –7 –7 Olfr876 Olfr1 –6 –6 Olfr983 and 45 (63%) responded. Of the 25 ORs enriched Of 25 the ORs enriched responded. and 45 (63%)

EN Olfr376 9 –5 –5 Olfr1238 C Control Acetophenone (1% –4 –4 Olfr1104 E

–3 –3 Supplementary Table 2 Supplementary ) Olfr1 + pS Olfr57 in vivo in advance online publication online advance Olfr196 6 9 Response Olfr744 0.1 0.2 0.3 0.4 0.5 0.6 Response Olfr166 0 0 1 2 3 4 5 6 Olfr875 , we performed pS6 staining along along staining pS6 performed we , – – ∞ ∞ log log P d Olfr1333 Olfr1054 Olfr1094 Olfr6 Olfr491 Olfr746 Olfr887 Olfr875 Olfr6 Olfr110 ) –9 –9 < 0.05), 24 were tested tested were 24 0.05), < + 10 Fraction of pS6 cells 10 0.2 0.4 0.6 0.8 1.0 [acetophenone] (M) [acetophenone] (M) 2 0 –8 –8 in vivo in 0 –7 –7 1 0 –6 –6 Olfr19 and and −9 % –5 –5 100% to 10 a Read counts 1,000 ) Left, read counts of acetophenone receptors in control and 1% acetophenone–stimulated acetophenone–stimulated 1% and control in receptors acetophenone of counts read ) Left, –4 –4 ). ). We that reasoned 200 400 600 800 in vitro in –3 –3 0 0.2 0.4 0.6 0.8 1.0 Olfr376 −3 0

Fig. 4a Fig. Olfr19

M acetophe Olfr749

P Response 1 0 0.1 0.2 0.3 Response

< 0.05), 71 Olfr923 Olfr923 evidence, evidence,

0 0 1 2 3 4 Olfr30 – – % Olfr1047 in vitro in ∞ ∞ log log , 100% b Olfr1444 Olfr1009 Olfr133 Olfr490 Olfr1477 Olfr376 Olfr136 Olfr430 Olfr1093 Olfr983 Olfr136 –9 –9 10 ). Of Of ). 10 Olfr1104 [acetophenone] (M) Dorsal [acetophenone] (M) –8 –8 Olfr744 1.0 0.2 0.4 0.6 0.8 - 0 –7

–7 Olfr110

Olfr875 –6 –6 1 0 observed for the 100% acetophenone stimulation ( stimulation acetophenone 100% the for observed enriched more be to tend −0.395; = rho ( stimulation 1% acetophenone following on change scales fold log transcript with for the 48 acetophenone ORs. Indeed, EC vitro in is not clear. the of we Toplotted OR sensitivity question, this address late well, the extent to which the pS6 method captures the information ( OR control the with compared as ORs acetophenone identified ing express OSNs in induction pS6 in resulted exposure acetophenone no showed that OR control a and ORs acetophenone identified newly the of six for RNA fluorescent with

Olfr1104 Olfr895 Fig. 4c Fig. –5 –5 Olfr490 Although the binary responses of responses binary the Although % Olfr874 –4 –4 100% Olfr876 –3 –3

EC Olfr983 ,

d Olfr1477 0.2 0.4 0.6 0.8 1.0 and and Olfr1463 50 0 Response Olfr1448 values against the the against values 0.05 0.10 0.15 Response P Olfr1377 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 0 Supplementary Fig. 3 Fig. Supplementary Fig. 5 Fig. = 0.005, linear regression and ANOVA, Spearman’sANOVA, and regression linear 0.005, = Olfr1444 0 0 Olfr430 in vitro in – –

% Olfr160 ∞ ∞ log log Olfr1009 Olfr1448 Olfr3 Olfr876 Olfr895 Olfr1463 Olfr502 Olfr920 Olfr4 Olfr191 Olfr1238 100% –9 –9 10 10 a Olfr935 [acetophenone] (M ), suggesting that ORs more sensitive sensitive more ORs that suggesting ), [acetophenone] (M) 0 4 –8 –8 Olfr133 n situ in response or enrichment in pS6-IP. Indeed, Indeed, pS6-IP. in enrichment or response 0.2 0.4 0.6 0.8 1.0 –7 –7 Olfr491 0 Olfr1333 in vivo. in –6 –6 Olfr1054 1 0 Olfr1094 Olfr736 –5 –5 hybridization using specific probes probes specific using hybridization n in vivo in

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µ Olfr746 % – – in in vitro m. ( m. ∞ ∞ log log Olfr904 100% Olfr736 pCI Olfr5 Olfr196 Olfr923 Olfr160 Olfr749 Olfr166 Olfr904 –9 –9 10 Olfr736 t r a 10 d [acetophenone] (M) [acetophenone] (M) P –8 –8 7 Olfr60 ) Quantification ) Quantification responses of responses 0.2 0.4 0.6 0.8 1.0 = 0.993, linear linear 0.993, = Olfr145 0 –7 –7 results corre results Olfr1132 (control) Olfr887 Olfr1484 –6 –6 ) 1 0 Olfr1093 C I –5 –5 in vitro in % –4 –4 e l 100% –3 –3

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© 2015 Nature America, Inc. All rights reserved. enriched or enriched only by 100% acetophenone ( acetophenone 100% by only enriched or enriched not ORs with compared as ORs sensitive more of fraction higher a contain to tended acetophenone 1% by enriched ORs of set the this, with Consistent stimulation. odor high-intensity following OSNs in pathways signaling olfactory the of saturation of result a as sumably 0.00137; = rho Spearman’s ANOVA, and regression acetophenone. 1% by enriched those with compared as acetophenone 100% by enriched ( acetophenone. 1% by enriched and acetophenone 100% by pS6-IP,in enriched only enriched not ORs of (log sensitive not and −4) and −2 between (log sensitive more ( = 0.00137. rho Spearman’s experiment pS6-IP the in stimulation acetophenone 100% following abundance transcript acetophenone for measured values ( = −0.395. rho Spearman’s ANOVA, and experiment pS6-IP the in stimulation acetophenone 1% following abundance transcript RNA of change fold and ( 5 Figure t r a  odorant concentrations better preserve information regarding OR OR regarding information preserve better concentrations odorant is for useful capturing the maximum number of ORs, responding lower concentrations odorant high with pS6-IP although that, suggest significant ( statistically not were results these although 100%, by enriched only those of that than lower be to tended acetophenone 1% by enriched test; Chi-square ORs have greater overall homology with each other. Toeach with surprise, our homology overall greater have ORs Having a large set of ORs, acetophenone we these next asked whether ORs acetophenone of relationship Sequence-function odorant. the to sensitivity d a

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© 2015 Nature America, Inc. All rights reserved. ORs activated by both acetophenone and TMT. and ( acetophenone both by activated ORs TMT, by represents red and activated ORs represents blue acetophenone, by activated ORs represents Orange ORs. TMT and acetophenone comparing ( (right). group acetophenone–stimulated 7 Figure regression logistic using built was model second A acetophenone. to responding not and responding ORs the separates best that space parameter the in surface hyper community the finds algorithm SVM learning the Briefly, machine the in used commonly as kernel, basis radial with (SVM) machine vector support the using built was significant no both acetophenone showed to response that ORs 367 and as receptors identified we acetophenone that ORs 48 the contained schemes. modeling for validation data The external and cross-validation tenfold both in them tested and approaches different two took we acetophenone, to To build models linking OR protein sequences to their responsiveness ( of one in them present mainly were ORs acetophenone the identified four clusters using the first three principal components, and a principal component analysis on the amino acid properties to information predict whether or not a sufficient given OR responds to acetophenone. Indeed, contain may sequences protein primary binding. odorant in implicated helices transmembrane in conserved among ORs as highly a whole not and the are majority of sites them are located these Notably, details). for Methods Online see n ( chance random by expected be would than ORs Similarity Elastic net SVM external validation Table 1 nature nature (blue, receptors a b = 17, 17, =

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–log (P value) P 0 5 10 in vivo in 10 12 value Fig. 6 Fig. 0.1 0 2 4 6 8 – ∞ −120 Olfr180 Olfr527 Olfr171 Olfr463 Olfr449 – −97 –9 – 6 P P b <0.05 <0.001 log b ) ) and and log – 4 and and –8 In vitro In 10 2 [TMT] (M) (fold change) 0 2 –7 d ( in vitro in n ( Supplementary Fig. 4 Fig. Supplementary ) Amino acid residues that were conserved in the acetophenone receptors (orange, (orange, receptors acetophenone the in conserved were that residues acid ) Amino =4) n a –6 =2) 0.66 0.716 0.716 responses of the TMT receptors. Error bars indicate s.e.m. ( s.e.m. indicate bars Error receptors. TMT the of responses ) Volcano plot showing enrichment of OR mRNA in the 100% TMT–stimulated group (left) and 1% and (left) group TMT–stimulated 100% the in mRNA OR of enrichment showing plot ) Volcano AUC External validation 4 2 –5 n . The first model model first The . –4 = 44, 44, = 6

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[TMT] (M –7 ping protocol to identify ORs responsible for detecting TMT TMT for detecting responsible ORs to identify protocol ping mice in predator-naive responses TMT,odor the chose fear-related a that induces fox component feces We clear. not is odorants other for used be can method the whether We used acetophenone to comprehensively identify OR repertoire, but ORs TMT of Identification P ( significance statistical with to acetophenone responsiveness time. In contrast, the overall similarity based approach did not predict the of 71.6–81.5% acetophenone to responsiveness predicting rectly P models showed predictive values in significant both cross ( expression functional of failures as such factors confounding exclude to successfully system heterologous our in expressed functionally were ORs human These rate data set for external validation of models ( acetophenone response profiles of 27 human ORs and the used this sepa obtained also we addition, In scheme. cross-validation tenfold a in tested were models the of All responses. OR predict similarity to sequence overall the using classifier a implemented also we As a comparison, over-fitting. to reduce introduced was step tion net penalty elastic using selection variable with Table 2 Table evidence that these 21 ORs are indeed TMT receptors ( expressed when TMT to cloned 42 of these ORs, and 21 of them displayed significant responses 43 100% TMT-enriched ORs ( 43 ORs ( OE ( the dissected We stimulated mice with 100% TMT for 1 h and performed pS6-IP on = 0.1; 0.1; = = 3 × 10 × 3 = –6 ) –5 ). Somewhat unexpectedly, six of the TMT ORs were also also were ORs TMT the of six unexpectedly, Somewhat ). –4 Fig. 6d Fig. P < 1% 4 0.05). TMT ORs enriched ( Acetophenone andTMT TM Acetophenone −97 Response T ) and external ( external and ) 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0 , e and and – n ∞ = of4 led to TMT stimulation pairs). enrichment Olfr110 Olfr57 Olfr30 Olfr96 Olfr181 –9 Table log –8 10 in vitro in [TMT] (M) in vitro in –7 1 P ). Fig. Fig. 7 –6 d c = 0.04, 0.04, = ) Distance tree of OR protein sequences sequences protein OR of tree ) Distance 1 –5 32 2 ( . Indeed, the SVM and elastic net net elastic and SVM the Indeed, . Conserved inallORs and TMTORs Conserved amongbothacetophenone Conserved amongTMTOR Conserved amongacetophenoneORs , Fig. 7 Fig. a 3 –4 3 and . . We our OR map applied next P

Response cor and validation 0.04) = Supplementary Fig. Supplementary 6 b –0.05 0.05 0.10 0.15 0.20 ). Thus, we found strong strong found we Thus, ). P 0 Supplementary Supplementary Fig. 5 < a of 0.05), subset the – 3 ∞ 1 . The variable . selec The variable pCl Olfr464 P t r a –9 < 0.05), TMT TMT < 0.05), log Supplementary –8 10 s [TMT] (M) –7 C I P = 10 –6 in vitro in P in in vivo e l = 0.1, –5 ). ). We −120 –4 s ). ).  - - - - . ,

© 2015 Nature America, Inc. All rights reserved. kinetics underlying these two methods, as well as method-specific method-specific as well as methods, two these underlying and kinetics pathways signaling different conditions, stimulation different Weid der of von findings the support data our random; than better no performs P Seq predicted the ORs identified by von der Weid RNA- pS6-IP in enrichment that revealed curve ROC an generating the ( set of data our one in criteria least at by supported were ORs (95%) 22 of 21 Overall, showed ORs (88%) 17 of 15 addition, In tration. ( enriched significantly were ORs (81%) 18 of which concentration, one at least change fold log positive showed ORs of 20 (91%) 22 data. expression Weid der von by Wescripts. the set examined of ORs acetophenone ( tran OR of downregulation than faster much occurs induction pS6 whereas OE, RNA from whole with working requires only and steps isobutyrate ethyl and acetophenone to responding ORs identify to stimulation advantage of the in decrease OR abundance transcript following odor using researchers enable by of ORs odorants activated interest. to identify species may non-model which animals, transgenic specific require not does pS6-IP addition, In time). stimulation odor of h 14 S100a5-tauGFP, pS6 induction showed faster onset (30 min to the 1 large-scaleh versusidentification of ORs for a given odor. As compared with enables ORs the method that our are pS6-IP whereas most the active, invitro both by supported muscone, for ORs two and eugenol GFP sorted of analysis array reporter the using S100a5 ORs to drive GFP expression in deorphanize activated OSNs, followed to by micro assay Kentucky the called method a reported study recent A patterns. odor- of activation OR view induced global a providing greatly while experiment, throughput one the in enhancing ORs mouse expressed all, not if most, screens pS6-IP Second, relevant. are more physiologically to be likely performed in awake, freely behaving animals; thus, the results obtained tages over the currently available techniques. First, experiments can be advan few a has method pS6-IP respectively.OR-odorant The pairs, Applying this approach to acetophenone and TMT identified 48 and 21 ORs mammalian odor-activated comprehensivelyidentify to are limited to several well-studied ORs. We developed a new approach proteins ORs the fluorescent with of interest ands are ORs which activated but ORs, of subset certain a activates odorant One DISCUSSION odorants. diverse for ORs active identify to used be can RNA-Seq with combination in pS6-IP that Together, suggest data our behaviors. of TMT-induced basis the These ORs represent candidates for future investigation to understand fear-related behavioral responses of TMT were in expressed the dorsal OE, a region implicated in the mediating ( binding ligand Five of and Olfr376 TMT in Olfr491) these Olfr57, ORs Olfr30, (Olfr20, involved been have may and helices, served residues among TMT ORs were also located in transmembrane con the ORs, acetophenone among residues conserved of majority ( sequence in diverse also were ORs TMT the ORs, acetophenone to Similar receptors. acetophenone as identified t r a  to contribute likely receptors, positive defining for cutoffs statistical

= 4 × 10 In this issue, Von der Weid der Von issue, this In in vivo methods. The Kentucky assay may be suitable for identifying for identifying suitable Kentucky may be The assay methods. C I in vivo in −9 P 3 < 0.05, FDR adjusted for 22 ORs) at least in one concen one in at least ORs) for 22 adjusted FDR < 0.05, , , Wilcoxon test, one rank-sum against H tailed has been facilitated by transgenic animals that coexpress that coexpress animals by transgenic facilitated been has 4 e l . As compared with pS6-IP, this method involves fewer fewer involves method pS6-IP, this with compared As . s is largely unclear. Although the search for OR lig OR for search the Although unclear. largely is et al. et 3 4 Supplementary Table 3 Supplementary in our pS6-IP RNA-Seq and heterologous heterologous and RNA-Seq pS6-IP our in + cells et al. et Supplementary Fig. 7 Fig. Supplementary 2 4 . Three ORs were identified for identified were ORs Three . 3 4 report a method that takes takes that method a report 3 3 ( Supplementary Fig. Supplementary 6e 8 , these animal resources resources animal , these Fig. 7 Fig. et al. et ). Quantification by Quantification ). et al. in vitro in n 3 c 4 = 22) identified ). Similar to the the to Similar ). 3 as groups. The The groups. as 4 (AUC = 0.86, ). Together, ). in vivo in activation. activation. 0 : classifier : classifier Fig. 7 Fig. in vivo in and and d ). ). ). ). ------.

may not fully capture the biological significance. Increasing the the Increasing significance. biological the capture fully not may used enrichment and activation of definitions statistical the Finally, odor. the to responded OR coexpressed the if enriched be may ORs non-responding case which in OSNs levels, some high at ORs that multiple express possibility the exclude cannot also one unlikely, ORs of some expression functional poor is sibility ORs additional activate and mucus nasal the in are into chemicals converted other is that molecules odor differences these for explanation possible One future. the in addressing worth between are there discrepancies some by ORs a activated Nonetheless, to odorant. given support determine ligand, given a of receptors. high-affinity for saturate may levels concentrations the higher are with levels associated phosphorylation usually higher Although S6. of levels rylation phospho OR influence for each affinities ligand and concentrations tion of intensity signal pS6 that suggests immunostaining both ligand the pathway signaling to leading Our S6 quantifica phosphorylation. identify to necessary be will study Future S6. of phosphorylation to in vitro methods. two the of one in identified only were ORs certain the differences in outcome. Future research is to necessary clarify why to their respective activating odors, the majority of which were unique diverse, we residues that identified are in similar the two of types ORs markedly. vary may odorants by different activated ORs tuned and broadly tuned rowly ORs few a activate only may Unlike acetophenone and TMT, some odors. of range odors, expanded an such with future the as in muscone, determined be could however, which tuned, broadly are ORs these that possibility the raising odor ants, both by activated were ORs Six respectively. ORs, 21 and 48 perception. and detection odor in ORs active the of each of roles the address to studies future ( mice behaving freely in concentration odor higher a by activated ORs additional as well as ORs sensitive more of identities the revealed odors of concentration cases in some quality odor perceived the affect may but intensity, odor ceived sensitivity. OR of regardless ligand of concentration high the by saturated are pathways signaling olfactory the neurons, It concentration. that, stimulation is for high possible many olfactory between correlation see not did we change contrast, In also interactions. OR-odorant of may kinetics mucus the nasal the in proteins odorant-binding of existence the Furthermore, vary. can to exposed is OR each tration concen odorant exact the epithelium, olfactory the in pattern zonal mucus nasal the in dissolved tion can airflow,influence which in turn can affect the odorant concentra correlation. For instance, the anatomical structure of the nasal passage the between Differences concentration. at a stimulation low enrichment interest. of odor an for ORs active identifying for strategy powerful a represents method our shortcomings, potential frequently chosen and expressed at low levels in the OE. Despite these for ORs less hypothesis null the against test might to better more reads give sample) per reads ~10,000,000 (currently depth sequencing We established a stringent criteria requiring both between correlations high found we general, In Although Although the of overall sequences acetophenone and TMT ORs are of set large a activated TMT and acetophenone that found We per the increase only not odor given a of concentrations Higher We found good correlation between in vivo in mapping results, strongly suggesting that OR activation leads leads that OR activation suggesting strongly mapping results, advance online publication online advance and and 3 8 . Our unbiased mapping of ORs activated at different different at activated ORs of mapping unbiased Our . in vitro in in vitro in systems may help to explain the noise in this this in noise the explain to help may systems Figs. 2 2 Figs. sensitivities and and sensitivities 15 , 2 4 and 3 . Following with the logic of nar of logic the with Following . 7 . Because ORs are expressed in a in expressed are ORs Because . 4 ,

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, nature nature 39 and sensitivities and in vivo in , 4 0 , the number of ORs , number the 35 in in vitro in vivo in in vitro , 3 enrichment at a at enrichment 6 NEUR . Another pos Another . n vivo in OR activity OR activity and . Although . Although OSCI in vitro in vivo EN and and C E ------

© 2015 Nature America, Inc. All rights reserved. 5. 4. 3. 2. 1. reprints/index.htm at online available is information permissions and Reprints The authors declare no competing interests.financial and H.M. wrote the paper. of the OR constructs. Y.J. and R.P. performed pS6-IP. Y.J. performed bioinformatics and statistical analysis. M.J.N. cloned some H.M. experiments. performed immunohistochemistry Y.J. and H.M. performed all of H.M. the supervised experiments and data analysis. N.N.G., Y.J., X.S.H. and US National Institutes of Health and (DC012095 DC014423). and comments on the manuscript. This work was supported by grants from the and sequencing S. service, Mukherjee and J. Mainland for helpful discussions Genome Sequencing and Analysis Core Resource for providing Illumina of the OR constructs, D. Marchuk for sharing of equipment, the Duke IGSP We thank G. andBarnea R. Axel for antibodies to M72, H. Zhuang for some online version of the pape Note: Any Supplementary Information and Source Data files are available in the at GEO: Accession codes. the in versio available are references associated any and Methods M coding. information olfactory of mapping odors is understanding for to likely deeper additional guide OR animals. behaving awake, in stimuli odor ORs specific to identify responsive comprehensively and efficiently to us enable idation RNA-Seq, ‘onethe pS6-IP and rule receptor’ neuron-one interact. ORs and how odors plement chemoinformatics studies ligand-based com will studies informatics receptor-based Our ligand. odor same the of question how can with ORs the interact with structures diverse help to answer should studies modeling molecular lacking, currently accommodate the same molecule. As information structural of ORs is could pockets binding unrelated multiple hand, other the On ferent. dif are sequences overall the though even pocket binding similar a form by odor same the is that ORs activated possibility One ORs. the of selectivity ligand to contribute indirectly or directly may residues acetophenone based on its primary sequence information. The shared to responds OR given a not or whether independent predict both that approaches two using models developed we responsiveness, and protein ace OR sequence to between Torelationship the tophenone. pursue OR further responsiveness that their about suggests information contain sequences sequences OR on component performed principal analysis Furthermore, binding. ligand in implicated domains transmembrane the at located are residues conserved these to and non-overlapping the ORs between of each tested odor. Most of nature nature COM AUTH Acknowledgmen

et In summary, odor-induced S6 phosphorylation in activated OSNs, in activated In S6 phosphorylation summary, odor-induced Peterlin, Z., Firestein, S. & Rogers, M.E. The state of the art of odorant receptor odorant of art the of state The M.E. Rogers, & S. Firestein, Z., Peterlin, a by coding Odor J.D. Mainland, & H. Matsunami, H., Zhuang, Q., Chi, H., Saito, Malnic, B., Hirono, J., Sato, T. & Buck, L.B. Combinatorial receptor codes for odors. receptor olfactory the of expansion Extreme Touhara,K. & A. Y., a Matsui, Niimura, receptors: odorant encode may family multigene novel A R. Axel, & L. Buck, erhnzto: rpr fo te orphanage. (2014). the from report a deorphanization: repertoire. receptor mammalian Cell mammals. placental 13 in groups gene orthologous of dynamics evolutionary and elephants African in repertoire gene recognition. odor for basis molecular P h O n of the pape the of n

E ods 96 TI R R NEUR GSE59 , 713–723 (1999). 713–723 , CON NG FI TRIBUTI OSCI N l . 32 A Raw reads and quantification results can be accessed NC 4 . r EN ts . r IA . C ON L E I

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© 2015 Nature America, Inc. All rights reserved. 37 °C for 15 min then 80 °C for 15 min. 5 1× PCR buffer (Roche), 1.5 mM MgCl 1 min. 10 for °C 65 then min 10 for °C 37 3 0.4 U 1 min and cooled to 4 °C. 0.3 0.4 U U 0.04 TTT), TTT TTT TTT TTT TTT TTT ng 2 with 5 described as generationsequencing. c (Qiagen). Chemicals were purchased from Sigma if not specified otherwise. with 350 temperature. After removing supernatant, RNA was eluted by mixing the beads During the final wash, beads were placed onto the magnet and moved to room beta-glycerophosphate).mMpyrophosphate, 1sodium mM orthovanadate,1 (Promega),100 7.4 adjusted with KOH), 1% NP-40, 2 mM DTT, 100 U ml (RNase-free water containing 350 mM KCl, 5 mM MgCl the RNA-bound beads were washed four times with 700 incubation,rotation. After with °C 4 at h 1 forincubation byfollowed mixed, After antibody binding, the mixture was added to the washed beads and gently adjusted7.4 (pHHEPES KOH),withmM 10 (wt/vol),NP-40).BSA0.05% 1% washed three times with 900 100 (Invitrogen, Dynabeads A Protein binding, antibody rotation.During with °C 4 at h 1.5 formixture the incubatingby allowed was 20 with mixedand for 10 min at 4 °C. The supernatant was transferred to a new 1.5-ml lobind tube, DHPC (Avanti Polar Lipids) were added. The mixture was centrifuged ata new17,000 1.5-ml lobind tube, to which 90 and centrifuged (Eppendorf), at 2,000 tube lobind 1.5-ml a to transferred was homogenate The and homogenized three times at 250 rpm and nine times at 750 ml)) rpm (Glas-Col).10 per tablet 1 (Roche, inhibitorbeta-glycerophosphate,protease mM 1 orthovanadate,sodium mM fluoride,pyrophosphate,1sodium mM 1 A, 2 mM DTT, 100 U ml KCl, 5 mM MgCl ice. on The beta-glycerophosphate) dissected OE mM was transferred 1 to 1.35 pyrophosphate,ml of homogenization sodium buffer (150 mM mM 1 date, 100 Ca (Gibco,HBSS buffer(1×withdissection of ml 25 in dissected wasOE theand cantly decreased the RNA yield. Following odor stimulation, lowpS6 background thein the OE, and pilot mouseexperiments with the peptide signifi was killed signal-noise ratio was not included Knightin by our used protocolwas that peptide because240–containing pS6 weThe observed relatively pS6-IP from the were littermates of the same sex. quadruplicates,and within each replication theexperimental and control mice triplicatesin orwereExperimentssolutionperformedcontrol odor orh. for1 the to exposed wasmousetransferred, the andalso was mouse the intowhich cassettea in (Tissue-Tek). Thecassette placedwasintomouse new containera or 10 rest for 1 h in an odorless environment. For odor stimulus, 10 vidually into sealed containers (volume O of Duke University. harvesting were approved by the Institutional Animal Care and Use Committee were used at the age of 3-4 weeks. The procedures of animal handling and tissue Animals. ONLINE MET nature nature U DN µ dor stimulation. µ 2+ g l −1 µ snhss P synthesis, A and Mg µ µ µ µ g ml g µ RNase H (Invitrogen), 1× PCR buffer (Roche), 3 mM dATP, 1.5 mM MgCl l µ l −1 l of distilled water (control) was applied to 1-cm × 1-cm filter paper held l l −1 −1 −1 l reaction mix (1× PCR buffer (Roche), 1.5 mM MgCl µ T4 gene 32 protein (Roche)) was added to each tube and incubated at WT C57BL/6 mice were purchased from Jackson. Animals of either sex poly-T primer (TAT AGA ATT CGC GGC CGC TCG CGA TTT TTT CGA TCG CGC GGC CGC ATT(TAT AGA primer poly-T recombinant RNasin (Promega)). This mixture was heated at 65 °C for NEUR RNase inhibitor (Qiagen), 4 U −1 l RLT (Qiagen). The eluted RNA was purified using RNeasy Micro kit cycloheximide, sodiumfluoride,mM 5 sodiumorthovanamM 1 9 2+ with modifications. Briefly, 1.5 1.5 Briefly, modifications. with ), 2.5 mM HEPES (pH 7.4 adjusted with KOH), 35 mM glucose, µ OSCI 2 H OE , 10 mM HEPES (pH 7.4 adjusted with KOH), 100 nM Calyculin g ml g 3–4-week-old C57BL/6 mice (Jackson Labs) were placed indi ODS g µ for 10 min at 4 °C. The supernatant was then transferred to . C l pS6 antibody (Cell Signaling, #2215). Antibodybinding#2215).Signaling, antibody (Cell pS6 l pS6-IP was conducted as described with modifications −1 EN apiiain n lbay rprto fr next- for preparation library and amplification R −1 cycloheximide, mMsodium5fluoride, mMsodium1 RT-PCR from asmallamount of RNA was conducted C RNasin (Promega), 100 E µ µ l beads wash buffer 1 (150 mM KCl, 5 mM MgCl l RT mix (170 U 2 µ ) was added to each tube and incubated at ≈ l 10% NP-40 (vol/vol) and 90 µ 2.7 l) inside a fume hood and allowed to l TdT mix (1.25 U µ l µ −1 l ExoI mix (2 U (2 mix ExoI l recombinant RNasin (Promega), µ µ l µ l −1 µ −1 l purified RNA was mixed mixed was RNA purified l g ml Superscript II (Invitrogen), RNase inhibitor(Qiagen), RNase −1 −1 µ 2 cycloheximide, 5 mM , 10 mM HEPES (pH l beads wash buffer 2 recombinant RNasin µ µ l et al. et −1 µ l per sample) was sample) per l µ l of odor solution 2 TdT (Roche), 0.1 , 50 l −1 1 ExoI (NEB), ExoI 7 to improve to µ µ M dNTPs, l 300 mM 1 2 7 2 g - - - ) . ,

distributed based on read counts from uniquely mapped reads. For example,wereregions twoif toa mapped Reads reads. mapped uniquely only using genes respectively. BEDTools was used to first count reads mapped to coding exons of separated into 20 sub-files that included reads mapped only to 1, 2, …, 20 20 differentregions regions on the reference genome), and the resulting mappingaligned tofile mm10 was by Bowtie with −m 20 −a (reports all reads that map to at most on previousthebased read counts from regionsthese reads aligned to multiple regions to be distributed proportionally to thesein multipleregions alignments, we implemented a stepwise mapping scheme that allows genes to count the mapped reads. Because the similarity between ORs the can all resultforexons coding coordinatesof the used only we defined, well not are were then counted using BEDTools reference genome mm10 using Bowtie Reads mapping and test for enrichment. quantification results can be accessed at GEO: libraries, 12 libraries per lane) in 50 base pair single read mode. Raw readsindexed and pooled (for 2000/2500 HiSeq or libraries) individual (for MiSeq on tionNexterawith DNA Sample Prep kits(Illumina). Libraries were sequenced prepara library for used was product purified the of ng 50 and purification, for each cycle, and then 72 °C for 10 min. The PCR product was purified by gel then 16 cycles of 95 °C for 30 s, 67 °C for 1 min, 72 (TaKaRa))°C for and incubated at3 95 °C for 2min min, 37 °C for 5 min,with 72 °C for 20 min, 6-s extension buffer (TaKaRa), 0.25 for mM dNTPs, °C 20 ng 65 then min 20 for 10 °C min. 3.5 37 at incubated and tube each to added was component that represents the non-responding group. When dose-response When group. non-responding the represents that component stimulation.cut-offchosensuchThewasthat it excluded Gaussian ofthe 99% 300 atincrease activityluciferase of fold the fit to used was model mixturetwo-componentGaussian A follows: as constructed was none The cut-off to determine positive or negative for (Olfr1126) 100. Data was analyzed using GraphPad Prism 5 or custom R scripts. controlpositiveand 0, was emptyvector with transfected cellsurements from For fold of induction, the fold of signal curves, increase background was dose-response the scaled so For that luciferase (Promega). meas protocols manufacturer’s following and Luciferase diluted in CD293 for 4 h at 37 and °CpRL-SV40. 18–24 andh later, 5%cells were COstimulated by incubation with odorants cells were transfected with plasmids coding for OR, M3-R, ouslyRTP1S, CRE-luciferase D and penicillin-streptomycin with (vol/vol) amphotericin B, at FBS 37 °C and 5% CO 10% containing (MEM) C known to facilitate the heterologous expression of ORs Rho-tag(firsta amino20 acids of rhodopsin) at N-termini.the The Rho-tagis verification. Unless otherwise noted, ORs were cloned into Rho-pCI to introduceAmplified fragments were cloned into pCI (Promega) or Rho-pCI for sequence fied using Phusion polymerase (Finnzymes) following manufacturer’s protocols. DN individual libraries OR group by controlling FDR the power of detection tions assumpmodelemploying similar DESeq2 toolseparate a usingobtained was analyzed using the negative-binomial model based tool EdgeR indicated that the majority of read counts stopped increasing after 20 cycles. then rounded. Iteration number 20 was chosen because pilot parameter tuning werecounts read resulted the and cycles 20 for allowed iterationwas The 7.7. 0.7 were added to the previous read counts and the new counts became 3.3 and were 3 and 7, respectively, based on uniquely mapped read counts, then 0.3 and read was mapped to two different genes, and the read counts of these two genes ell culture. ell ual- h ra cut tbe o al h gns ee hn motd no and R into imported then were genes the all for table counts read The A and vector preparation. vector and A 4 2 6 G 6 . Littermate information was included in the design matrix to enhance to matrix design the in included Littermateinformationwas . . Briefly, Hana3A cells were plated on 96-well plates. 18–24 h after plating, lo assay. µ l of the product was added to 27.5 aaA cells Hana3A Renilla The Dual-Glo assay for ORs was performed as described previ Renilla 4 6 . 4 luciferase activities were measured using Dual-Glo kit Dual-Glo using measured were activities luciferase 7 luciferase activity was subtracted from each data set. set. data each from subtracted was activity luciferase . Multiple comparison was adjusted within the detected 9 ee anand n iia esnil medium essential minimal in maintained were 4 8 The open reading frames of ORs were ampli were ORs of frames reading open The . DESeq was used to calculate the size factors of 4 3 2 . Because (to date) the UTRs of many ORs . 4 2 . The reads mapped to annotated genes µ Short reads were aligned to the mouse 2 l to allow for CRE-luciferase expression. −1 poly-T primer, 0.05 U GSE5932 µ l PCR mix (1× LA Taq reaction in vitro 4 2 4 3 4 . Briefly,. readsthe were responses to acetophe . . doi: µ 4 5 M acetophenoneM 10.1038/nn.4104 . A similar result µ l −1 LA Taq ------

© 2015 Nature America, Inc. All rights reserved. ojgtd nirbi IG Jcsn muooias 120 ws sd to used was 1:200) visualize pS6 protein signals. Immunologicals, (Jackson IgG Cy3- anti-rabbit donkey washing, conjugated Following °C. 4 at overnight 1:200) Signaling, (Cell tissues of 3-week-old mice were incubated with rabbit anti-phospho-S620- staining, (240/244) Immunohistochemistry and be functionally expressed in our heterologous system. grid search. External validation was performed using 27 human ORs that could forRBFkernel SVM; cut-offs to generate the ROC curves and AUC. Parameters ( possible True all atpositiveratestruth.werecalculated with false positiveand models were asked to predict the testing set and the predictions were compared regressionand modelsSVM were trained using trainingthe beforeset, trained sets, for 100 iterations: 90% as training subset and 10% as testing subset. Logistic performed on the mouse OR data. Each time, the data was divided into two sub was performed using the e1071 package. A tenfold cross-validation scheme was Elastic net logistic regression was performed using the glmnet package acetophenonerespondnotto didexperiments) andtion enriched in pS6 RNA-Seq ( and responded to acetophenone and both supportedwerebythat ORs To used responses,we 0–1 construct resulting from occasional gaps in alignment were imputed from column means. resulting in 3 × 291 = 873 predictors. The missing values in this matrix (0.17%) Grantham by defined (polarity,propertiesasvolume) compositionandacid amino lated Using the aforementioned multiple alignment of mouse OR proteins, we calcu acetophenone. to responsiveness predict to properties sequence OR using els residue frequencies was generated by WebLogo study. Multiple comparison was adjusted by controlling FDR sets. Locations with gaps for more than 10% of the ORs were excluded from the the null-hypothesis that they were samples from the distributions of random OR computed for the group of 48 acetophenone ORs, and used was iteratedto 20,000 times.find The per-residue p-values mean Grantham distancesunder were also these 48 ORs were calculated and their mean was computed. Random sampling repertoire. At each amino acid residue, the pairwise Grantham distances across these distributions, 48 random ORs were randomly sampled from the mouse OR distances within random OR sets for each residue in the alignment. To simulate none ORs than by chance, we first simulated the distributions of adjustments.mean Grantham To identify residues that were more conserved among disruptedthe acetophe were excluded from the analysis) wereregionstransmembrane predictedaligned more or by one Clustalxwith (pseudogenes withsequences manual acetophenone of analysis sequence Protein for stimulation. using 10 experimentsadditional by amplitude responseconfirmedindependently were small estimatedwithmaximumtheORs responses ofabove 0.intervals dence positive defined we rangewide a with ofstimulation curves concentrations were available ( doi: SVM and logistic regression with elastic net penalty were used to build mod 10.1038/nn.4104 invitro −5.5 2 9 evidence, with the48ORs that were enriched inour pS6 RNA-Seq , 10 for 291 amino acid residues common to at least 10% of the ORs, the of 10% least at to commonresidues acid amino 291 for µ m frozen sections of the olfactory epithelium and surrounding and epithelium olfactory the of sections frozen m −5 , 10 in vitro in −4.5 α = 0.07 for0.07= elastic netlogisticregression) were tuned by , 10 P −4 > 0.05 in both 1% and 100% acetophenone stimula responses as the lower bounds of the 95% confi 95% the of bounds lower the as responses in situ , 10 in vitro −3.5 hybridizations. , and 10 labeled as 1’s, and 367 ORs that were not −3 M acetophenone concentrations 4 O 9 . Rs. For immunohistochemical 1,090 mouse OR protein OR mouse 1,090 in vitroin γ 4 = 0.001, cost = 10 8 . Visualization of labeled as 0’s.as labeled 5 Fig. 0 in vivo in . SVM 4 ), ------

50. 49. 48. 47. 46. 45. The corrected total cell fluorescence was calculated as for used measuring the average fluorescence of the entire olfactory epithelium. maximumintensitya mergedasprojection, was and signal pS6 tification.The among the Z-stacks for the selected OR area was used for signal intensity quan yield an to integratedunits square densityarbitrary in value.area the The by maximummultiplied was integratedarea OR densityselected a measurementof manually. For each slice in a Z-stacked image, the average intensity of the pixels selected was area OR each ImageJ,and in projection intensitymaximum a as merged was signal OR the acquisition, Following bisbenzimide. for #49 and forforfluorescein,Cy3,#38#43 set follows:filterwere asZeiss used sets filter functionality to take optical sections that reduced background fluorescence. The Zeiss Axiocam MRm and upright inverted fluorescent microscope2- with ApoTome was performed as described above. fluorescein fluorophore. inductionthe using (PerkinElmer) aspS6Following forstainingFISH, (TSA) signals amplification signal Hybridization tyramide using (Roche). detected were DIG against antibody (HRP)-conjugated lium. After washing, the sections were incubated with a horseradish peroxidase 58°C to target mRNAs in 20- genin (DIG)-labeled complementary RNA probes were hybridized overnight at situIn the OR of interest, followed by immunohistochemical staining for pS6 induction. 44. 43. 42. cell × background signal) epithelium µ Background signal = average signal per pixel for the entire olfactory olfactory entire the for pixel per signal average = signal Background Whole-cell signal = maximum integrated density of a cell with images Z-stacked follows. as quantified were intensities signal pS6 Fluorescent A selected of (area – signal Whole-cell = fluorescence cell total Corrected

m intervals between each slice, were obtained at 200× magnification using the remn J, ate T & isiai R Rglrzto pts for paths Regularization R. Tibshirani, & T. Hastie, J., Friedman, logo sequence a WebLogo:Brenner, S.E. & J.M. Chandonia, G., Hon, G.E., Crooks, and practical a rate: discovery false the Controlling Y. Hochberg, & Y. Benjamini, McCarthy, D.J., Chen, Y. & Smyth, G.K. Differential expression analysis of multifactor data. count sequence for analysis expression Differential W. Huber, & S. Anders, for package Bioconductor a edgeR: G.K. Smyth, & McCarthy,D.J. M.D., Robinson, otzv, . Wlim, .. MCe K, cafe, . Wl, . apn and Mapping B. Wold, & L. Schaeffer, K., McCue, B.A., Williams, for comparing A., Mortazavi, of utilities suite flexible a BEDTools: I.M. Hall, & A.R. Quinlan, Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient eeaie lna mdl va oriae descent. coordinate via (2010). models linear generalized generator. 57 testing. multiple to approach powerful (2012). 4288–4297 variation. biological to respect with experiments RNA-Seq Biol. Genome (2010). 139–140 data. expression gene digital of analysis expression differential (2008). RNA-Seq. by transcriptomes mammalian quantifying features. genomic (2009). alignment of short DNA sequences to the human genome. human the to sequences DNA short of alignment Supplementary , 289–300 (1995). 289–300 , hybridization was carried out as previously described previouslyas out carried hybridizationwas Genome Res. Genome in situ

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