N treated with 30 ng/mL 5-AzaC for 72 hours prior to preparing histones.Methyl-DNA ImmunoprecipitationMeDIP was performed as described [42,43]. Briefly, DNA prepared from v6.5 and Eed2/2 cells at passage 13 and 6, respectively, was treated with RNase A, incubated at 37uC for 30 minutes, sonicated on a Covaris sonicator and isopropanol precipitated. 4 ug Sonicated DNA was incubated at 4uC overnight with 10 ug anti-5-MeC order CASIN antibody (Eurogentec, BI-MECY-0100) in IP buffer (10 mM Na-Phosphate pH7, 140 mM NaCl, 0.05 Triton). Samples were then incubated with anti-mouse M-280 Dynabeads (Invitrogen, 112.01D) for 2 hours in 0.1 BSA/PBS at 4uC. Samples were washed twice, treated with Proteinase K, extracted with 1:1 phenol/chloroform ethanol precipitated. 100 ng of each sample was then amplified using the GenomePlex Complete Whole Genome Amplification kit (Sigma, WGA2) and hybridized to a NimbleGen 36720 K CpG Island Plus RefSeq Promoter Array (NimbleGen, 05924537001). 20,404 promoters are on the array. The Cornell Life Sciences Core Laboratory performed array hybridization. Peaks were called using the NimbleGen Nimblescan software with the default settings. We defined a gene promoter as the region from 22 kb to +1 kb of a transcriptional start site according to the Ensembl annotation of the mm9 version of the mouse genome.Bisulfite Sequencing of PCR FragmentsBisulfite conversion was performed using the MethylEasy Xceed kit (Human Genetic Signatures, ME002). PCR products were gel purified and cloned using the TOPO TA Cloning Kit (Invitrogen, 45?641). Primers used are listed in Supplementary Table 4. At least 15 independent clones were sequenced and Homatropine methobromide biological activity analyzed using the QUMA platform (http://quma.cdb.riken.jp/).Cluster Analysis and Binning of Microarray DataClustering of microarray data was done using Cluster 3.0 [44]. Microarray data were reformatted for the Cluster program using custom perl scripts. Data from all three arrays were intersected using BEDTools [45] and binned using R. Data were visualized using Origin.ChIP-seq2615 cm dishes of ES cells were fixed in 1 formaldehyde/ PBS for 30 minutes at room temperature. Fixing was quenched with 1.4 mL 2.5 M Glycine. Cells were scraped into a 15 mL tube, washed twice with PBS and stored at 280uC. Chromatin was prepared by incubating cells 10 minutes at 4uC in lysis buffer 1 (50 mM Hepes, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10 Glycerol, 0.5 NP-40, 0.25 Triton w/protease inhibitors)DNAme and H3K27me3 in Mouse Embryonic Stem Cellsfollowed by a 20-minute incubation at room temperature in lysis buffer 2 (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris pH 8 w/protease inhibitors). Cells were resuspended in 500 uL buffer 3 (1 mM EDTA, 0.5 mM EGTA, 10 mM Tris pH 8 w/protease inhibitors) and sonicated on a Misonix 3000 sonicator for 90 seconds (30 seconds on/30 seconds off). 350 uL of sonicated chromatin was used to create ChIP-seq libraries as previously described [46].set of methylation data is a fisher’s exact test that was conducted for each CpG (* p,.05, ** p,.01, *** p,.001). Genes validated are a, Tdrd5, b, Pdgfrl, c, Gas5, d, Fbxo18, e, f, Fv1. Note that Fv1 is a gene with peaks of both increased (e) and decreased (f) DNAme in the promoter. (EPS)Figure S3 Validation of genes called as differentially expressed in DnmtTKO and Eed2/2 cells by RNAseq. a, RPKM values calculated by Cufflinks for genes used in qRT-PCR in (b). A box has been placed around all the genes called as significantly differentially.N treated with 30 ng/mL 5-AzaC for 72 hours prior to preparing histones.Methyl-DNA ImmunoprecipitationMeDIP was performed as described [42,43]. Briefly, DNA prepared from v6.5 and Eed2/2 cells at passage 13 and 6, respectively, was treated with RNase A, incubated at 37uC for 30 minutes, sonicated on a Covaris sonicator and isopropanol precipitated. 4 ug Sonicated DNA was incubated at 4uC overnight with 10 ug anti-5-MeC antibody (Eurogentec, BI-MECY-0100) in IP buffer (10 mM Na-Phosphate pH7, 140 mM NaCl, 0.05 Triton). Samples were then incubated with anti-mouse M-280 Dynabeads (Invitrogen, 112.01D) for 2 hours in 0.1 BSA/PBS at 4uC. Samples were washed twice, treated with Proteinase K, extracted with 1:1 phenol/chloroform ethanol precipitated. 100 ng of each sample was then amplified using the GenomePlex Complete Whole Genome Amplification kit (Sigma, WGA2) and hybridized to a NimbleGen 36720 K CpG Island Plus RefSeq Promoter Array (NimbleGen, 05924537001). 20,404 promoters are on the array. The Cornell Life Sciences Core Laboratory performed array hybridization. Peaks were called using the NimbleGen Nimblescan software with the default settings. We defined a gene promoter as the region from 22 kb to +1 kb of a transcriptional start site according to the Ensembl annotation of the mm9 version of the mouse genome.Bisulfite Sequencing of PCR FragmentsBisulfite conversion was performed using the MethylEasy Xceed kit (Human Genetic Signatures, ME002). PCR products were gel purified and cloned using the TOPO TA Cloning Kit (Invitrogen, 45?641). Primers used are listed in Supplementary Table 4. At least 15 independent clones were sequenced and analyzed using the QUMA platform (http://quma.cdb.riken.jp/).Cluster Analysis and Binning of Microarray DataClustering of microarray data was done using Cluster 3.0 [44]. Microarray data were reformatted for the Cluster program using custom perl scripts. Data from all three arrays were intersected using BEDTools [45] and binned using R. Data were visualized using Origin.ChIP-seq2615 cm dishes of ES cells were fixed in 1 formaldehyde/ PBS for 30 minutes at room temperature. Fixing was quenched with 1.4 mL 2.5 M Glycine. Cells were scraped into a 15 mL tube, washed twice with PBS and stored at 280uC. Chromatin was prepared by incubating cells 10 minutes at 4uC in lysis buffer 1 (50 mM Hepes, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10 Glycerol, 0.5 NP-40, 0.25 Triton w/protease inhibitors)DNAme and H3K27me3 in Mouse Embryonic Stem Cellsfollowed by a 20-minute incubation at room temperature in lysis buffer 2 (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris pH 8 w/protease inhibitors). Cells were resuspended in 500 uL buffer 3 (1 mM EDTA, 0.5 mM EGTA, 10 mM Tris pH 8 w/protease inhibitors) and sonicated on a Misonix 3000 sonicator for 90 seconds (30 seconds on/30 seconds off). 350 uL of sonicated chromatin was used to create ChIP-seq libraries as previously described [46].set of methylation data is a fisher’s exact test that was conducted for each CpG (* p,.05, ** p,.01, *** p,.001). Genes validated are a, Tdrd5, b, Pdgfrl, c, Gas5, d, Fbxo18, e, f, Fv1. Note that Fv1 is a gene with peaks of both increased (e) and decreased (f) DNAme in the promoter. (EPS)Figure S3 Validation of genes called as differentially expressed in DnmtTKO and Eed2/2 cells by RNAseq. a, RPKM values calculated by Cufflinks for genes used in qRT-PCR in (b). A box has been placed around all the genes called as significantly differentially.
