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1 SUPPLEMENTARY INFORMATION doi: /nature19360 Supplementary Tables Supplementary Table 1. Number of monoclonal reads in each sample Sample Number of cells Total reads Aligned reads Monoclonal reads H3K4me3_P12_rep H3K4me3_P12_rep H3K4me3_P15_rep H3K4me3_P15_rep H3K4me3_Oocyte_rep H3K4me3_Oocyte_rep H3K4me3_2-cell_rep H3K4me3_2-cell_rep H3K4me3_8-cell_rep H3K4me3_8-cell_rep H3K27ac_Oocyte_rep H3K27ac_Oocyte_rep H3K27ac_2-cell_rep H3K27ac_2-cell_rep H3K27ac_8-cell_rep H3K27ac_8-cell_rep Input_P12_rep Input_P12_rep Input_P15_rep Input_P15_rep Input_oocyte_rep Input_oocyte_rep Input_2cell_rep Input_2cell_rep Input_8cell_rep Input_8cell_rep
2 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Table 2. KAPA qpcr based quantification of ChIP-seq libraries to assess relative amounts of H3K4me3 ChIP precipitated DNA Sample Ct dct Log Relative Fraction amount Oocyte cell cell P P Supplementary Table 3. Summary of WGBS results from P12 and P15 Sample Bisulfite conversion rate Coverage of CpG sites Average CpG methylation Average read depth of CpG sites P12 99% 93% 19% 9.06 P15 99% 93% 25%
3 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Table 4. TF motif enrichment at distal cres nearby ZGA genes TF motif Consensus sequence P value RARg(NR)/ES-RARg-ChIP-Seq(GSE30538)/Homer AGGTCAAGGTCA RLR1?/SacCer-Promoters/Homer WTTTTCYYTTTT Nr5a2(NR)/mES-Nr5a2-ChIP-Seq/Homer BTCAAGGTCA CRX(Homeobox)/Retina-Crx-ChIP-Seq/Homer GCTAATCC Nr5a2(NR)/Pancreas-LRH1-ChIP-Seq(GSE34295)/Homer BTCAAGGTCA Pho2(bHLH)/Yeast-Pho2-ChIP-Seq(GSE29506)/Homer CCCACGTGCT c-myc/mes-cmyc-chip-seq/homer VVCCACGTGG n-myc(hlh)/mes-nmyc-chip-seq/homer VRCCACGTGG PIF5ox(bHLH)/Arabidopsis-PIF5ox-ChIP-Seq(GSE35062)/Homer BCACGTGVDN ZNF143 STAF(Zf)/CUTLL-ZNF143-ChIP-Seq(GSE29600)/Homer ATTTCCCAGVAKSCY ARE(NR)/LNCAP-AR-ChIP-Seq(GSE27824)/Homer RGRACASNSTGTYCYB Pitx1(Homeobox)/Chicken-Pitx1-ChIP-Seq(GSE38910)/Homer TAATCCCN Max(HLH)/K562-Max-ChIP-Seq/Homer RCCACGTGGYYN Rfx5(HTH)/GM12878-Rfx5-ChIP-Seq(GSE31477)/Homer SCCTAGCAACAG Erra(NR)/HepG2-Erra-ChIP-Seq/Homer CAAAGGTCAG BORIS(Zf)/K562-CTCFL-ChIP-Seq/Homer CNNBRGCGCCCCCTGSTGGC Bcl6(Zf)/Liver-Bcl6-ChIP-Seq(GSE31578)/Homer NNNCTTTCCAGGAAA PR(NR)/T47D-PR-ChIP-Seq(GSE31130)/Homer VAGRACAKNCTGTBC GFY-Staf/Promoters/Homer RACTACAATTCCCAGAAKGC GATA-DR8(Zf)/iTreg-Gata3-ChIP-Seq(GSE20898)/Homer AGATSTNDNNDSAGATAASN Esrrb(NR)/mES-Esrrb-ChIP-Seq/Homer KTGACCTTGA Gfi1b(Zf)/HPC7-Gfi1b-ChIP-Seq/Homer MAATCACTGC FOXA1:AR/LNCAP-AR-ChIP-Seq(GSE27824)/Homer AGTAAACAAAAAAGAACAND AR-halfsite(NR)/LNCaP-AR-ChIP-Seq(GSE27824)/Homer CCAGGAACAG FOXA1(Forkhead)/MCF7-FOXA1-ChIP-Seq/Homer WAAGTAAACA TR4(NR/DR1)/Hela-TR4-ChIP-Seq/Homer GAGGTCAAAGGTCA TATA-box/SacCer-Promoters/Homer BBHWTATATA MafK(bZIP)/C2C12-MafK-ChIP-Seq(GSE36030)/Homer GCTGASTCAGCA FOXA1(Forkhead)/LNCAP-FOXA1-ChIP-Seq(GSE27824)/Homer WAAGTAAACA p53(p53)/saos-p53-chip-seq/homer RRCATGYCYRGRCATGYYYN
4 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Table 5. Genome Ontology analysis of stage restricted putative distal cres GO biological process -log10 (P value) mesc-restricted distal cres Posttranscriptional regulation of gene expression Stem cell development Stem cell differentiation Cell junction organization Negative regulation of protein kinase activity Oocyte restricted distal cres Positive regulation of hormone biosyntheic process Regulation of hormone biosyntehic process Positive regulation of hormone metabolic process Regulation of lipid metabolic process Regulation of hormone metabolic process cell restricted distal cres Cell cycle Protein modification by small protein conjugation or removal M phase Cell cycle phase Cell division cell restricted distal cres Embryonic placenta development Plancenta development Regulation of circadian rhythm Negative regulation of glial cell differentiation Regulation of gliogenesis
5 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Table 6. Enriched mouse phenotypes of stage restricted putative distal cres Mouse phenotypes -log10(p value) mesc-restricted distal cres Decreased embryo size Abnormal embryo size Decreased erythrocyte cell number Small spleen Abnormal erythrocyte cell number Oocyte restricted distal cres Abnormal cell adhesion Abnormal bone volume Decreased percent body fat Abnpormal cell migration Abnormal endometrial gland morphology cell restricted distal cres Embryonic lethality between implantation and placentation Complete embryonic lethality before somite formation Embryonic lethality before somite formation Decreased cell proliferation Abnormal cell content or morphology cell restricted distal cres Long tail Abnormal allantois morphology Abnormal type II pneumocyte morphology Enlarged allantois Abnormal pulmonary alveoulu epithelial cell morphology
6 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Table 7. Enriched GO biological processes at nearby genes for each TF motif containing stage restricted putative distal cres Stage TF motif Enriched top 3 GO biological processes P value Jun-Ap1 negative regulation of signal transduction 7.4e-04 Jun-Ap1 negative regulation of cell communication 1.4e-03 Jun-Ap1 response to hormone stimulus 2.2e-03 BATCH2 negative regulation of signal transduction 1.2e-03 Oocyte BATCH2 intracellular signaling cascade 1.4e-03 BATCH2 actin filament-based process 1.5e-03 Nr5a2 enzyme linked receptor protein signaling pathway 1.5e-04 Nr5a2 fatty acid metabolic process 3.8e-04 Nr5a2 negative regulation of signal transduction 7.4e-04 cmyc protein transport 5.6e-09 cmyc establishment of protein localization 8.4e-09 cmyc protein localization 3.7e-08 RARg protein transport 2.0e-06 2-cell RARg macromolecule catabolic process 2.4e-06 RARg establishment of protein localization 2.7e-06 Rfx5 cell cycle 2.4e-11 Rfx5 RNA processing 1.3e-08 Rfx5 protein localization 2.7E-07 RARg translation 1.2E-10 RARg cell cycle 4.6E-10 RARg RNA processing 5.2E-10 RLR1 RNA processing 3.6E-15 RLR1 cell cycle 3.5E-14 RLR1 macromolecule catabolic process 8.4E-14 Nr5a2 macromolecule catabolic process 3.7E-12 ZGA Nr5a2 cellular macromolecule catabolic process 1.7E-11 Nr5a2 protein catabolic process 9.7E-11 cmyc RNA processing 5.0E-16 cmyc mrna metabolic process 1.1E-13 cmyc macromolecule catabolic process 1.2E-12 CRX RNA processing 6.4E-17 CRX cellular macromolecule catabolic process 8.6E-15 CRX macromolecule catabolic process 2.4E-14 RARg macromolecule catabolic process 3.2E-17 RARg cellular macromolecule catabolic process 4.6E-17 RARg cell cycle 2.6E-16 Pho2 macromolecule catabolic process 2.1E-13 2/8-cell Pho2 cellular macromolecule catabolic process 3.9E-13 Pho2 protein catabolic process 4.7E-12 Nr5a2 cellular macromolecule catabolic process 2.3E-17 Nr5a2 macromolecule catabolic process 4.4E-17 Nr5a2 cell cycle 1.4E-15 RARg phosphorus metabolic process 2.0E-03 RARg phosphate metabolic process 2.0E-03 RARg protein transport 2.9E-03 p53 heart morphogenesis 2.8E-02 8-cell p53 heart development 4.4E-02 p53 muscle tissue morphogenesis 6.2E-02 TEAD4 protein transport 2.2E-05 TEAD4 establishment of protein localization 2.7E-05 TEAD4 protein localization 4.4E-05 Oct4-Sox2-TCF- tube development 7.9E-04 ES-cell Nanog Oct4-Sox2-TCF- tube morphogenesis 3.0E
7 SUPPLEMENTARY INFORMATION RESEARCH Nanog Oct4-Sox2-TCF- positive regulation of macromolecule metabolic process 3.2E-03 Nanog p53 tolerance induction 1.3E-02 p53 cell division 5.9E-02 Sox2 tube development 2.5E-03 Sox2 establishment of cell polarity 2.6E-03 Sox2 vesicle-mediated transport 2.6E-03 Supplementary Table 8. RNA-seq of Kdm5a and 5b morpholino targeted 2-cell embryos and various controls Sample # of embryos Replicate Library Type Uniquely mapped reads Uninjected 25 rep1 Paired-end Uninjected 25 rep2 Paired-end α-amanitin 25 rep1 Paired-end α-amanitin 25 rep2 Paired-end Kdm5a+b MO 25 rep1 Paired-end Kdm5a+b MO 25 rep2 Paired-end Control MO 25 rep1 Paired-end Control MO 25 rep2 Paired-end Supplementary Table 9. PANTHER Overrepresentation Test for downregulated genes after Kdm5a+b MO injection PANTHER GO-slim biological process Cell-cell signaling Transport Metabolic process P value 1.5E-3 2.6E-2 2.0E-2 7
8 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 1. Original Simple Western immunoblot images for Kdm5a and Kdm5b morpholino knock-down of KDM5A and KDM5B proteins in 2-cell embryos. Beta-actin is shown as loading control. 8
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