Supplementary figures and tables

A 100 kb hg38

[0 - 10.00] GM_Pol 2_50K_1_Full _Norma10 lized _Extended .bwR1 50,000- 0 [0 - 10.00] GMnative_Pol 2_50K_2_Full _Norma10 lized _Extended .bwR2 0 [0 - 10.00] GM_Pol 2_10K_1_Full _Norma10 10,000lized _Extended-.bwR1 0 [0 - 10.00] GMnative_Pol 2_10K_2_Full _Norma10 lized _Extended .bwR2 0 [0 - 10.00] GM_Pol2_2K_1_Full _Normal10 ized2,000_Extended-.bw R1 0 [0 - 10.00] GMnative_Pol 2_2K_2_Full _Normal10 ized _Extended .bw R2 0 [0 - 10.00] GM_Pol 2_1K_1_Full _Normal10 ized1,000_Extended-.bw R1 0 [0 - 10.00] GMnative_Pol 2_1K_2_Full _Normal10 ized _Extended .bw R2 0 [02 - 2.00] CL-Yan090 ...tended .bw 100,000- R1 0 [02 - 2.00] crosslinkedCL-Yan090 ...tended .bw R2 0 ENCODE_GM_Pol 2-4H8_1_10Ful[0 - 10.00] l_NormalizedENCODE_Extended .bw 0

RefSeqGene gene ZC3H10 MYL6MYL6 RNF41RNF41 ANKRD52 CS CNPY2 IL23AIL23A TIMELESSTIMELESS SPRYD4 SNORA105C RBMS2RBMS2 RBMS2 BAZ2ABAZ2A PTGES3PTGES3 NACANACA HSD17B6

Correlation of promoters Pol 2-S5 B 1.0 ENCODER2_R2 ENCODE ENCODER1_R1

0.9 X50KR2_R2 50,000- native X50KR1_R1

X10KR2_R2 10,000- 0.8 native X10KR1_R1

X2KR2_R2 2,000- native 0.7 X2KR1_R1

X1KR2_R2 1,000- native X1KR1_R1 0.6

FixR2_R2 100,000- crosslinked FixR1_R1

0.5 1 2 1 2 1 2 1 2 1 2 1 2 R R R R R R R R R R R R ______R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 K K K K K K K K 1 1 2 2 Fix Fix 10 10 50 50 - - - - - ENCODE ENCODE native native native native 1,000 2,000 10,000 50,000 100,000 ENCODE crosslinked

Supplementary Fig S1. Comparison of MNase-based native MOWChIP-seq with crosslinked MOWChIP-seq. (A) Normalized Pol II (S5) signal of GM12878 using MOWChIP- seq, with either native (1,000-50,000 cells per assay) or crosslinked (100,000 cells per assay) chromatin. (B) Pearson’s correlation matrix of native and crosslinked MOWChIP-seq data and ENCODE data (SRX100530), computed using DiffBind affinity score method.

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A 100 kb hg38 [0 - 10.00] GM_Pol 2_10K_1_Full _Norma 10 lized _Extended .bw R1 0 10,000 [0 - 10.00] GM_Pol 2_10K_2_Full _Norma 10 lized _Extended .bw R2 0 [0 - 10.00] GM_Pol 2_10K_st_1_Full _No 10 RNA Pol II (S5) rmalized _Extended .bw R1 0 [0 - 10.00] 10,000GM_Pol2_10-Kst_st_2_Full _No 10 rmalized _Extended .bw R2 0 [0 - 10.00] ENCODE_GM_Pol 2-4H8_1_Ful10 l_Normalized _Extended .bw ENCODE 0 Gene RefSeq geneZC3H10 MYL6MYL6 RNF41RNF41 ANKRD52 CSCS CNPY2 IL23AIL23A TIMELESSTIMELESS SPRYD4 SNORA105C RBMS2RBMS2 RBMS2 BAZ2ABAZ2A PTGES3PTGES3 NACANACA HSD17B6 B Correlation of promoters Pol 2-S5 11.0.0

XR210K_R2 00.9.9 10,000

X10K_R1 R1 00.8.8

0.7 X10KR2_st_R2 0.7 10,000-st 00.6.6 X10KR1_st_R1

00.5.5 1 2 1 2 R R R R _ _ _ _ K K st st _ _ R1 R2 R1 R2 10 10 K K 10 10 st - 0,000 1 10,000 Supplementary Fig S2. Storage at -80 °C does not change RNA pol II binding profile. (A) Normalized Pol II-S5 signal of GM12878 cells. “10,000-st” denotes chromatin samples (each containing chromatin fragments from 10,000 cells) that were stored at -80 °C for 2 d and then used for nMOWChIP-seq. “10,000” refers to samples processed without such storage. In contrast, we found that freezing of chromatin samples at -80 °C substantially varies TF binding profiles. (B) Pearson’s correlation matrix of Pol II-S5 nMOWChIP-seq data of fresh and -80 °C stored GM12878 chromatin, computed using DiffBind affinity score method.

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A B C

753 443 855

2312 4635 1564

45673

PEGR1FC_EGR1 PMEF2CFC_MEF2C PPolFC _IIP-ototall2-total Supplementary Fig S3. Overlap among Pol2-total, EGR1 and MEF2C peaks of GM12878 cells. Peaks were identified by MACS2 using default settings. Peaks from both replicates that overlap more than 50% of their length are merged into a common peak using bedtools. The overlap of the common peaks was plotted using Intervene.

380 123 22

812

3097 38

3

30356 [0 - 2.00]

CLu-JE8-P...tended .bw

[0 - 2.00]

CLu-JE8-P...tended .bw A 100 kb mm10 [0 - 2.00] CLu-JE8-PFC-HDAC2-mix -10 2 CLuK-1_-JES188-_PL...006tended_R1_.bw001_Full R1 _Normalized _Extended .bw 0 [0 - 2.00] PFCCLu mixed-JE8-PFC-HDAC 100K2-mix -10 2 CLuK-2_-JES198-_PL...006tended_R1_.bw001 _Full R2 _Normalized _Extended .bw 0 [0 - 2.00] CLu-JE8-PFC-HDAC2-mix -10 2 KCLu-1_-SJE188-_PL...006tended_R1_.bw001_Full R1 _Normalized _Extended .bw 0 PFC NeuN+ 80K[0 - 2.00] CLu-JE8-PFC-HDAC2-mix -10 2 KCLu-2_-SJE198-_PL...006tended_R1_.bw001 _Full R2 _Normalized _Extended .bw 0 [0 - 2.00] Refseq genesRefSeq gene CLu-JE8-P...tended .bw Gal3st3 Cst6 Sart1Sart1 Drap1Drap1 Fibp Cfl1Cfl1 Ovol1Ovol1 Rnaseh2c RelaRela Pcnx3 Ehbp1l1Ehbp1l1 Scyl1 Malat1Malat1 Neat1 Slc25a45Slc25a45 Pola2Pola2 Capn1Capn1 Syvn1Syvn1 Zfpl1 Snx15Snx15 Batf2

[0 - 2.00]

CLu-JE8-P...tended .bw Correlation of promoters TERC B 1.01.0

R2100K_2 PFC mixed 0.90.9 100K

100K_1 R1 0.80.8

0.7 R280K_2 0.7 PFC NeuN+ 80K 0.60.6 R180K_1

0.50.5 1 2 1 2 _ _ _ _ K K K K 80 80 100 100 R1 R2 R1 R2 + 80K 100K NeuN PFC mixed mixed PFC PFC PFC Supplementary Fig S4. HDAC2 binding in mouse PFC samples profiled by nMOWChIP- seq. (A) Normalized HDAC2 signals generated using nuclei from mouse PFC. 100,000 unsorted nuclei from PFC, and 80,000 of FACS-sorted NeuN+ (neuronal) nuclei were used per assay in these tests. (B) Pearson’s correlation matrix on HDAC2 mouse PFC data, computed using DiffBind affinity score method.

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GM12878

15%

39%

37%

9%

No Pol II binding Paused and unexpressed (PI>=20) Paused and expressed (2<=PI<20) Non-paused and expressed (PI<2) Supplementary Fig S5. Distribution of pausing index (PI) for genes in GM12878 cells based on Pol II-total nMOWChIP-seq data.

5

A

PPFCFC_EEGR1GR1 PPFCFC_MMEF2CEF2C PPFCFC_PPolol2-IIto-totatal l

B

380 123 22

812 PCerebellumFC_EGR1 EGR1 3097 38 PCerebellumFC_MEF2C MEF2C PCerebellumFC_Pol2-tot Polal II-total

Supplementary Fig S6. Overlap among Pol II, EGR1, and MEF2C binding peaks in mouse PFC (A) and cerebellum (B) data. Peaks were identified by MACS2 using default settings. Peaks from both replicates that overlap more than 50% of their length are merged into a common peak using bedtools. The overlap of the common peaks was plotted using Intervene.

30356

6 380 123 22

812

3097 38

30356 Supplementary Table S1. Summary of nMOWChIP-seq data Mapped Mapping Unique Unique File MACS2 FRIP reads Rate reads rate GM12878_Pol2-total_50K_1 16432424 98% 16204517 99% 133015 57.1% GM12878_Pol2-total_50K_2 20219467 98% 19820603 98% 129219 55.5% GM12878_Pol2-S5_50K_1 16006273 98% 15570715 97% 142339 52.4% GM12878_Pol2-S5_50K_2 15203135 98% 14377380 95% 130669 47.3% GM12878_Pol2-S5_10K_1 12271356 96% 12061753 98% 114811 41.3% GM12878_Pol2-S5_10K_2 21780356 96% 21550914 99% 140695 48.0% GM12878_Pol2-S5_10K_st_1 9646207 95% 9354458 97% 82320 27.8% GM12878_Pol2-S5_10K_st_2 13775137 96% 13338787 97% 79800 24.9% GM12878_Pol2-S5_2K_1 14243331 90% 13432201 94% 59713 17.7% GM12878_Pol2-S5_2K_2 15242929 87% 14400694 94% 70439 22.0% GM12878_Pol2-S5_1K_1 15572762 87% 14923018 96% 33627 10.7% GM12878_Pol2-S5_1K_2 9194036 86% 8777057 95% 19820 8.1% GM12878_Pol2-S5_100K_cl_1 31745569 98% 31406005 99% 53817 8.8% GM12878_Pol2-S5_100K_cl_2 31111394 98% 30545134 98% 17561 2.7% GM12878_EGR1_100K_1 14323523 96% 12199195 85% 16614 8.3% GM12878_EGR1_100K_2 17458334 96% 14961426 86% 15584 7.9% GM12878_EGR1_50K_1 14910367 95% 12800282 86% 16465 8.6% GM12878_EGR1_50K_2 16669698 95% 14480504 87% 15159 8.2% GM12878_EGR1_10K_1 9072849 84% 7725182 85% 2626 3.8% GM12878_EGR1_10K_2 12461029 94% 10785293 87% 2781 2.6% GM12878_EGR1_5K_1 10330104 92% 8383719 81% 8245 5.0% GM12878_EGR1_5K_2 12660454 93% 11175451 88% 1423 1.9% GM12878_MEF2C_100K_1 24369737 76% 21133894 87% 14716 4.5% GM12878_MEF2C_100K_2 24642860 85% 20705206 84% 19884 6.3% GM12878_HDAC2_100K_1 21068312 84% 18801502 89% 11221 3.8% GM12878_HDAC2_100K_2 19862691 85% 18127467 91% 5832 2.3% GM12878_HDAC2_50K_1 8767036 93% 8093468 92% 1204 1.3% GM12878_HDAC2_50K_2 12215492 93% 11358639 93% 1584 1.1% PFC_Pol2-total_50K_1 13064208 97% 12803650 98% 66123 48.6% PFC_Pol2-total_50K_2 16395371 99% 16251965 99% 80138 55.4% PFC_Pol2-total_10K_1 8574476 95% 7748982 90% 24423 26.5% PFC_Pol2-total_10K_2 13007858 93% 12338679 95% 53291 46.4% Cerebellum_Pol2-total_50K_1 10617272 99% 10444570 98% 43387 52.0% Cerebellum_Pol2-total_50K_2 9805738 98% 9553592 97% 36762 48.5% Cerebellum_Pol2-total_10K_1 15483210 97% 15200342 98% 54534 67.1% Cerebellum_Pol2-total_10K_2 14308273 98% 14139656 99% 54278 69.5% PFC_EGR1_100K_1 9285737 91% 8104052 87% 6212 10.7% PFC_EGR1_100K_2 8557087 51% 7510446 88% 6921 11.0% Cerebellum_EGR1_100K_1 9882524 95% 8739064 88% 17795 16.2% Cerebellum_EGR1_100K_2 13141514 95% 11799729 90% 22866 15.4%

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PFC_MEF2C_100K_1 8478934 82% 7497951 88% 1354 7.5% PFC_MEF2C_100K_2 11407867 91% 10012928 88% 5362 9.1% Cerebellum_MEF2C_100K_1 11268711 95% 10016208 89% 24063 19.0% Cerebellum_MEF2C_100K_2 11481247 95% 10025093 87% 17393 15.6% PFC_HDAC2_100K_1 14011639 85% 11164030 80% 2929 13.6% PFC_HDAC2_100K_2 15354891 85% 12429080 81% 3465 12.6% PFC_NeuN+_HDAC2_80K_1 12258720 80% 10188120 83% 1382 11.2% PFC_NeuN+_HDAC2_80K_2 12448796 80% 10639071 85% 2842 9.6%

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Supplementary Table S2. List of genes with significant difference in pausing index between PFC and cerebellum based on mouse brain Pol II-total data (fold change > 3, minimal read density > 0.02) PFC < Cerebellum PFC > Cerebellum

Igfbp6 Dlk2 Plk2 Rps6ka1 Npas4 Mycl1 Rec8 Carhsp1 Snapc2 Shank3 Kcnf1 Sparcl1 Nsg1 Fgfbp3 Slc1a2 Slc16a8 Grik5 Gpsm3 Camk2n1 Smtn Sox12 Ankrd33b Paqr6 Fosb Dos Trip6 Chtf18 Flrt2 Sept5 Cul5 Tro Gm11696 Barhl2 Gp5 Gm2506 Cbln1 Syt7 E130303B06Rik Zfp296 Htr1b Gpr123 Fam101a Arpp19 Proca1 Nfyb Ccnt1 Rapgefl1 Pak6 Bex2 Gpt Lmo4 Lrg1 Arc Btbd17 Celf5 Pnrc1 Tubb2a Ascl1 Ephx4 Aqp5 Tmem151a Kank2 Nap1l1 Tcte1 Gpc1 Eif4e Ino80e 2610203C20Rik Gnmt Chrm4 Kif17 Ramp2 Emilin3 A230052G05Rik Mtch1 0610007L01Rik

9

Arpp21 Hrc Ptprn Doc2a Rgs2 Rsph9 Kcnt1 Npy1r Hrh3 Eno3 Stk19 Scn3b BC018242 Ntn3 Klhdc8a Ndrg4 A930018M24Rik Shroom1 Sun2 Dusp18 Prkag1 Loxl3 Pigz Ttc19 Atp2b1 Dnaja3 Sstr3 Def6 Zdhhc8 Plch2 Man2a2 Bdh1 Tlcd2 Nol3 Rgs4 Coch Ppp1r1a Gm20537 Micall1 Acta1 Rasd1 Tmem88b Eid2 Cox6b2 Tacc1 Fam164c Rtn3 BC106179 Ncan Fosl1 Bai2 S1pr5 Gm996 Ntpcr Gria2 Msl3l2 Trim9 Arpc1b Arhgdia Slc16a3 Nrn1 Pqlc3 Srm 1700019N12Rik Vipr1 Set Gm10116 Kank3 Fam49a Mad2l2 Gm4535 Acyp1 Ttyh3 BC025920 Gnao1 Ltk Ywhaz Xrcc3 Tceb2

10

Sh3glb2 1700088E04Rik Camk2n2 Arpc2 C030046I01Rik Cacnb1 1110008P14Rik Gal3st3 Usp30 Ccdc157 Dpysl2 Trim46 Atp1a3 R3hdm1 Eme1 Aifm3 Asphd1 Mcl1 Ppil2 Josd1 Cdc42ep3 Mcrs1 Bloc1s2 Rhog Klhdc8b BC017643 Rnf139 Jdp2 4931428F04Rik Slc4a3 Rabggta Psenen Efhd2 Pcgf2 Rab35 1700019L03Rik Hexdc Tcea2 Dusp7 Banf1 Ccdc103 Pdlim7 Pacsin3

11

Epn3 Sepw1

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Supplementary Table S3. Primers used in qPCR enrichment tests of nMOWChIP-seq libraries.

Species Locus name Primer Actg1 (F) CGG AAA GAT CGC CAT ATA TGG AC Actg1 (R) ACC GGC AGA GAA ACG CGA Polr2a (F) GAG AGA CAA ACT GCC GTA ACC Human Polr2a (R) GGG AAA TAA GGA GCG AAA GGA G (GM12878 cells) Afm (F) GCA GAA CCT AGT TCC TCC TTC AAC Afm (R) AGT CAT CCC TTC CTA CAG ACT GAG A Kctd16 (F) GCC ATA AAG AAA CAC ACA TGG AAA C Kctd16 (R) GCA AAG CAG GAA CCC TCA AA Arc (F) CAG CAT AAA TAG CCG CTG GT Arc (R) GTC GCC GCT GAA GCT AGA Polr2a (F) AAA GAA GGG AGG AGA GGA GGA Mouse brain Polr2a (R) GGG AGA GAC AAA CTG CCG TAA (PFC and Afm (F) CAT TTG ACC CAA ACT GCT AAG T cerebellum) Afm (R) GTA TGT GAA GTG TCA GCA ATG G Gcg (F) TCA ACC CAA AGT CCC TGA AG Gcg (R) CCA AGA GGT TGC ATT GGA AG

13