Supplementary Information for the Skeletal Muscle Molecular Clock Regulates Sarcomere Length Through Titin Splicing
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Supplementary Information for The skeletal muscle molecular clock regulates sarcomere length through titin splicing Lance A. Riley, Xiping Zhang, Joseph M. Mijares, David W. Hammers, , Hailey R. Olafson, Ping Du, Collin M. Douglas, Siegfried Labeit, Eric T. Wang, and Karyn A. Esser Karyn A. Esser Email: [email protected] This PDF file includes: Tables S1 to S7 1 Table S1. Antibodies and concentrations used in this study. Antibody Source Concentration Rb anti-sarcomeric α-actinin (EP2529Y) Abcam 1:1000 Rb anti-titin N2A Myomedix 1:250 Rb anti-titin Z1Z2 Myomedix 1:100 Rb anti-RBM20 Myomedix 1:500 Ms anti-γ-tubulin (T6557) Sigma 1:1000 Rt anti-HA High Affinity (11867423001) Roche 1:1000 Gt anti-rabbit Alexa Fluor 488 Thermo 1:500 Gt anti-rabbit Alexa Fluor 405 Thermo 1:500 Gt anti-rabbit Alexa Fluor 647 Thermo 1:500 Gt anti-rabbit IgG (H+L) HRP Sigma 1:10000 Gt anti-mouse IgG (H+L) HRP Sigma 1:10000 2 Table S2. RNA Integrity Values and number of reads sequenced for each sample used for RNAseq analysis. Sample Name RIN Read Number (Millions) M10 8.6 61.4 M11 8.3 58.6 M13 8.4 77.5 M15 8.9 69.3 M16 8.8 45.6 M17 8.7 59.8 3 Table S3. Primers used for plasmid generation and qRT-PCR. Sequence (5’ – 3’) Name GGGAGATCTTTAACAACATAGGAGCTGTGATTGGCTGT U7-BglII F AAACTGCAGCACAACGCGTTTCCTAGGAAACCA U7-PstI R GCTCTTTTAGAATTTTTGGAGCAGGTTTTCTGAC SDM U7smOpt F GTCAGAAAACCTGCTGGTTAAATTCTAAAAGAGC SDM U7smOpt R TTAGGGTGGGTGGATACGCCTCTGCAAAAGAATTTTTGGAGCAGGTTTTCTG Ttn-51-AS F TATCCACCCACCCTAAGTCCCTATCATAGCGGAAGTGCGTCTGTAG Ttn-51-AS R TAGGGTGCAAGGTACTCCTTAGAGTGAAAGAATTTTTGGAGCAGGTTT Ttn-89-AS F GTACCTTGCACCCTAAGTCCCTATCATAGCGGAAGTGCGTCTGTAG Ttn-89-AS R TGCATGCCCAGAAATGCCTGCT Rbm20 qPCR F AAAGGCCCTCGTTGGAATGGCT Rbm20 qPCR R CGAGTCCAGCGAGAGAAGG Rpl26 F GCAGTCTTTAATGAAAGCCGTG Rpl26 R CTAGGACGGAATCTGCTGTG Rbm20 Intron 1 F AACAGGGTGTCTGTCTGTCT Rbm20 Intron 1 R 4 Table S4. Differentially expressed genes in iMSBmal1-/- muscle as assessed by RNAseq. log2 fold Gene Name P-value Padj-value change Gm38158 -2.690632567 5.26E-08 8.68E-06 Gm26908 -2.211605045 4.62E-16 2.29E-13 Gm45819 -2.204676046 1.90E-18 1.65E-15 Gm29724 -2.150367038 4.52E-10 9.79E-08 SphK2 -1.820716149 2.66E-27 9.21E-24 Gm16268 -1.677681571 2.23E-08 3.86E-06 Gm27342 -1.650860415 1.12E-08 2.12E-06 0610040F04RiK -1.358121213 1.14E-15 4.92E-13 Gm45418 -1.348715562 4.07E-05 0.002477723 Gm14493 -1.3138007 1.36E-10 3.15E-08 Smpd5 -1.305408763 6.19E-05 0.003637123 Sh2d7 -1.261395179 3.12E-13 9.00E-11 Gm15824 -1.258159667 3.55E-11 8.80E-09 WnK2 -1.253674226 0.000397106 0.016192575 Aebp1 -1.228620787 0.001306931 0.040764384 MylK4 -1.225874401 0.000760075 0.027730723 PrKag2os2 -1.064535019 0.000268463 0.012406565 Gm11670 -1.014201233 0.001666204 0.04778543 Cd163 -1.009051298 1.16E-08 2.12E-06 Fggy -0.988749199 0.001091681 0.036480944 Usf3 -0.987594873 1.73E-06 0.000193382 Btf3l4 -0.964949379 2.40E-05 0.001568459 Thra -0.948845588 1.36E-13 4.28E-11 Gm37422 -0.901977283 6.98E-13 1.86E-10 Gm26584 -0.854012217 1.85E-05 0.001330976 Igsf3 -0.823314082 2.77E-05 0.001745923 A1bg -0.821055043 6.54E-05 0.003778259 Dtna -0.819934467 1.33E-05 0.00100373 Filip1l -0.813440092 0.000157009 0.008002862 Gm17034 -0.807531099 0.001538233 0.045568517 Teddm2 -0.790589866 6.01E-07 8.01E-05 Cyp27a1 -0.783739397 0.000525778 0.020475819 CerKl -0.748722569 2.69E-05 0.001723975 Gm48265 -0.746540211 0.001535447 0.045568517 Gm15477 -0.735319843 0.000105494 0.005480697 Gm11655 -0.71325289 0.000919437 0.031552152 Sorbs2os -0.708283397 0.001259414 0.040176721 Shisa3 -0.707744512 2.23E-05 0.001484356 Gm9860 -0.698409044 0.000310598 0.013626993 Phyhipl -0.684265458 0.001054037 0.035816578 Ccdc107 -0.656081901 2.32E-07 3.65E-05 5 Clcn2 -0.652899402 0.0004377 0.017239422 Gmpr -0.650803313 1.98E-05 0.001364209 1700037C18RiK -0.648333848 1.49E-06 0.000171747 Gm44085 -0.635390235 0.001422724 0.043638597 Gm48488 -0.631167401 0.001589209 0.046679633 Myhas -0.628745119 1.07E-05 0.000922918 Gm16740 -0.621468937 0.00024714 0.011575496 mt-Nd4 -0.617675897 1.27E-05 0.00100373 Tecpr2 -0.610707311 3.89E-07 5.40E-05 Paqr3 -0.596198997 0.000368097 0.015371371 Gm48752 -0.589799719 3.52E-07 5.30E-05 Platr31 -0.577672518 0.00029171 0.013130726 Gm15511 -0.574664889 0.001147976 0.037227567 Gm43909 -0.565823651 0.001094639 0.036480944 Gins2 -0.564494548 5.60E-06 0.000530187 Top2b -0.561864443 0.000182341 0.008777706 Baz2a -0.558497873 9.45E-05 0.005118951 A930028N01RiK -0.540776518 0.000105945 0.005480697 Gm26911 -0.540174896 0.000871775 0.030520947 Gm27241 -0.528189227 0.001663011 0.04778543 Gm26995 -0.523378635 1.30E-05 0.00100373 Gm26835 -0.51722508 0.000866564 0.030520947 Tbce -0.500057156 1.09E-05 0.000922918 Gm43034 -0.499332883 0.001488566 0.044864099 Ttc23 -0.493555167 3.63E-06 0.000380918 Gm47003 -0.492375046 0.000287218 0.013098671 Gm28289 -0.48599424 0.001263492 0.040176721 Gm4814 -0.484585079 4.47E-06 0.000442697 mt-Ty -0.471935074 5.66E-06 0.000530187 Gm28376 -0.46673907 9.25E-05 0.005087829 Atxn1 -0.466723783 0.000893085 0.030954334 4930506C21RiK -0.466283082 2.88E-05 0.00178308 1110046J04RiK -0.458401294 0.001317256 0.040764384 3110021N24RiK -0.452789396 0.000220297 0.010459589 Gm45102 -0.452398508 4.36E-06 0.000442697 Gm15910 -0.428119987 0.00042568 0.016958718 Yipf2 -0.417340139 0.000310565 0.013626993 B430010I23RiK -0.413978991 0.000357627 0.015116285 A630019I02RiK -0.403362288 1.82E-05 0.001330976 Appl1 -0.395848085 0.000160907 0.008082651 Tnnt3 0.343513392 0.001149264 0.037227567 Neb 0.356268872 0.000597759 0.022767389 Gm37759 0.371797797 0.000401826 0.016194533 Mrpl47 0.375323024 0.000756519 0.027730723 6 CKm 0.402012771 0.001460165 0.044394147 Igsf8 0.445729145 9.42E-06 0.000836979 Snw1 0.466303701 0.001308396 0.040764384 Acta1 0.472681086 0.000609599 0.022965969 Nr1d1 0.554090434 6.69E-05 0.003799817 Myh2 0.556656012 0.001733727 0.049254909 Prss23os 0.57462031 2.10E-06 0.000226966 Gm16214 0.574907568 0.000825698 0.029778629 Gm48008 0.601335764 0.001145994 0.037227567 Gm26631 0.602704435 1.43E-09 2.91E-07 Cyb5r2 0.603401649 1.88E-05 0.001330976 Gm37804 0.622513299 0.00016444 0.00814215 Atp1a2 0.681624806 0.000173115 0.008450935 4430402I18RiK 0.697996003 0.000394156 0.016192575 Slc9a2 0.739759433 0.001668216 0.04778543 A730049H05RiK 0.753036801 0.000833389 0.029778629 Arhgef17 0.762703024 7.39E-06 0.000673972 Mylpf 0.819114069 7.55E-07 9.69E-05 Gm26892 0.852820812 1.24E-05 0.00100373 Pvalb 0.872121786 9.69E-05 0.005166672 DapK2 0.939177898 0.000318581 0.013802502 HipK3 0.980105085 8.20E-05 0.004582232 Zcchc17 1.00895635 1.31E-13 4.28E-11 Hectd2 1.010732256 3.88E-07 5.40E-05 Rab11b 1.04239438 2.01E-05 0.001364209 Plet1 1.195856863 4.92E-05 0.00293757 P2ry6 1.241381939 0.000700101 0.026091939 1700071M16RiK 1.31102399 8.28E-15 3.19E-12 AlpK2 1.468990328 1.40E-06 0.00016786 Gm36235 1.610275442 0.000339959 0.014546898 Adora1 2.29729709 1.33E-05 0.00100373 Vps13c 2.510055807 1.39E-19 1.61E-16 Mboat1 2.627965513 4.25E-17 2.62E-14 Prss51 3.177668501 2.34E-26 4.05E-23 Gm44787 3.962987956 4.53E-17 2.62E-14 7 Table S5. Panther gene ontology analysis results on genes containing a significant splicing change in iMSBmal1-/- muscle. GO Biological Process Fold Enrichment P-value FDR striated muscle myosin thick filament assembly 18.42 1.84E-04 2.91E-02 skeletal muscle myosin thick filament assembly 18.42 1.84E-04 2.88E-02 myosin filament assembly 16.58 2.53E-04 3.77E-02 myosin filament organization 16.58 2.53E-04 3.73E-02 cardiac myofibril assembly 11.6 7.82E-06 3.06E-03 myofibril assembly 5.44 2.13E-04 3.28E-02 cardiac cell development 4.91 1.76E-04 2.87E-02 cellular component assembly involved in 4.27 2.21E-04 3.35E-02 morphogenesis actomyosin structure organization 4.02 3.44E-04 4.85E-02 protein modification by small protein removal 3.95 1.07E-04 2.02E-02 spindle organization 3.89 1.23E-04 2.11E-02 peptidyl-lysine modification 3.17 3.46E-05 9.51E-03 muscle cell differentiation 3.03 3.85E-05 1.01E-02 striated muscle cell differentiation 2.95 3.24E-04 4.62E-02 muscle system process 2.85 2.99E-04 4.29E-02 muscle structure development 2.54 1.53E-05 5.34E-03 supramolecular fiber organization 2.43 7.01E-05 1.55E-02 modification-dependent macromolecule 2.41 3.82E-05 1.01E-02 catabolic process modification-dependent protein catabolic 2.38 6.52E-05 1.50E-02 process protein modification by small protein 2.36 1.63E-05 5.31E-03 conjugation or removal ubiquitin-dependent protein catabolic process 2.35 1.05E-04 2.01E-02 peptidyl-amino acid modification 2.34 2.39E-06 1.10E-03 actin cytoskeleton organization 2.33 1.15E-04 2.06E-02 heart development 2.31 3.89E-05 9.83E-03 proteolysis involved in cellular protein catabolic 2.31 5.52E-05 1.29E-02 process protein catabolic process 2.3 1.56E-05 5.29E-03 cellular protein catabolic process 2.23 7.93E-05 1.66E-02 actin filament-based process 2.23 1.46E-04 2.43E-02 macromolecule catabolic process 2.19 4.73E-06 1.90E-03 circulatory system development 2.18 1.38E-06 7.18E-04 chromatin organization 2.17 8.93E-05 1.75E-02 cellular macromolecule catabolic process 2.12 4.10E-05 1.00E-02 regulation of cellular response to stress 2.12 8.89E-05 1.76E-02 cytoskeleton organization 2.06 3.16E-06 1.34E-03 organonitrogen compound catabolic process 2.06 1.56E-05 5.19E-03 8 Table S6.