ERS3905267 SAMEA6104586 10090 Mus musculus Protocols: Cells were collected by trypsinization To differentiate naïve ESCs into Epiblast-like cells (EpiLCs), flasks were first pre-treated with Geltrex (Life Technologies) diluted 1:100 in DMEM/F12 (Life Technologies) and incubated at 37 °C overnight. Naïve ESCs were plated on Geltrex-treated flasks in defined medium containing 10 ng/mL Fgf2 (R&D Systems), 20 ng/mL Activin A (R&D Systems) and 0.1× Knockout Serum Replacement (KSR) (Life Technologies). Media was changed after 24 h and EpiLCs were harvested for RRBS and RNA-seq experiments after 48 h. For the generation of Tet1, Tet2, and Tet1/Tet2 catalytic mutants, specific gRNAs targeting the catalytic center of Tet1 and Tet2 were cloned into a modified version of the SpCas9-T2A-GFP/gRNA (px458;(Ran et al., 2013), Addgene plasmid #48138), to which we fused a truncated form of human Geminin (hGem) to SpCas9 in order to increase homology-directed repair efficiency (Gutschner et al., 2016). A 200 bp ssDNA oligonucleotide harboring the H1652Y and D1654A mutations and ~100 bp of homology to the genomic locus was synthesized (IDT, Coralville, IA, USA). For targetings in wild-type J1 ESCs, cells were transfected with a 4:1 ratio of donor oligo and Cas9/gRNA construct. Positively transfected cells were isolated based on GFP expression using fluorescence-activated cell sorting (FACS) and plated at clonal density in ESC media 2 days after transfection. Cell lysis in 96-well plates, PCR on lysates, and restriction digests were performed as previously described (Mulholland et al., 2015). Tet1 catalytic mutation was confirmed by Sanger sequencing. For insertion of the HALO or eGFP coding sequence into the endogenous Dppa3 and Uhrf1 loci, respectively, Dppa3 and Uhrf1 specific gRNAs were cloned into SpCas9-hGem-T2A-Puromycin/gRNA vector, which is a modified version of SpCas9-T2A-Puromycin/gRNA vector (px459;(Ran et al., 2013), Addgene plasmid #62988) similar to that described above. To construct the homology donors plasmids, gBlocks (IDT, Coralville, IA, USA) were synthesized containing either the HALO or eGFP coding sequence flanked by homology arms with ~200-400 bp homology upstream and downstream of the gRNA target sequence at the Dppa3 or Uhrf1 locus, respectively, and then cloned into the NruI site of pUC57-GentR via cut-ligation. ESCs were transfected with equimolar amounts of gRNA and homology donor vectors. Two days after transfection, cells were plated at clonal density and subjected to a transient puromycin selection (1 ug/mL) for 40 h. After 5-6 days, ESCs positive for HALO or eGFP integration were isolated via fluorescence-activated cell sorting (FACS) and plated again at clonal density in ESC media. After 4-5 days, colonies were picked and plated on Optical bottom µClear 96-well plates and re-screened for the correct expression and localization of eGFP or HALO using live-cell spinning-disk confocal imaging. Cells were subsequently genotyped using the aforementioned cell lysis strategy and further validated by Sanger sequencing(Mulholland et al., 2015). Column purification For RRBS, genomic DNA was isolated using the QIAamp DNA Mini Kit (QIAGEN), after an overnight lysis and proteinase K treatment. RRBS library preparation was performed as described previously (Boyle et al. 2012), with the following differences: bisulfite treatment was performed using the EZ DNA Methylation-Gold™ Kit (Zymo Research Corporation) according to the manufacturer's protocol. ENA-FIRST-PUBLIC 2020-09-30T04:05:08Z ENA-LAST-UPDATE 2019-10-21T13:50:45Z External Id SAMEA6104586 INSDC center name Faculty of Biology Department II Ludwig Maximilians University Munich INSDC first public 2020-09-30T04:05:08Z INSDC last update 2019-10-21T13:50:45Z INSDC status public Submitter Id E-MTAB-6785:Tet1CM_EpiLC_RRBS_rep1 broker name ArrayExpress cell line J1 cell type epiblast-like cell common name house mouse developmental stage blastocyst disease normal genotype Tet1CM organism part inner cell mass sample name E-MTAB-6785:Tet1CM_EpiLC_RRBS_rep1 scientific_name Mus musculus ERS3905268 SAMEA6104587 10090 Mus musculus Protocols: Cells were collected by trypsinization To differentiate naïve ESCs into Epiblast-like cells (EpiLCs), flasks were first pre-treated with Geltrex (Life Technologies) diluted 1:100 in DMEM/F12 (Life Technologies) and incubated at 37 °C overnight. Naïve ESCs were plated on Geltrex-treated flasks in defined medium containing 10 ng/mL Fgf2 (R&D Systems), 20 ng/mL Activin A (R&D Systems) and 0.1× Knockout Serum Replacement (KSR) (Life Technologies). Media was changed after 24 h and EpiLCs were harvested for RRBS and RNA-seq experiments after 48 h. For the generation of Tet1, Tet2, and Tet1/Tet2 catalytic mutants, specific gRNAs targeting the catalytic center of Tet1 and Tet2 were cloned into a modified version of the SpCas9-T2A-GFP/gRNA (px458;(Ran et al., 2013), Addgene plasmid #48138), to which we fused a truncated form of human Geminin (hGem) to SpCas9 in order to increase homology-directed repair efficiency (Gutschner et al., 2016). A 200 bp ssDNA oligonucleotide harboring the H1652Y and D1654A mutations and ~100 bp of homology to the genomic locus was synthesized (IDT, Coralville, IA, USA). For targetings in wild-type J1 ESCs, cells were transfected with a 4:1 ratio of donor oligo and Cas9/gRNA construct. Positively transfected cells were isolated based on GFP expression using fluorescence-activated cell sorting (FACS) and plated at clonal density in ESC media 2 days after transfection. Cell lysis in 96-well plates, PCR on lysates, and restriction digests were performed as previously described (Mulholland et al., 2015). Tet1 catalytic mutation was confirmed by Sanger sequencing. For insertion of the HALO or eGFP coding sequence into the endogenous Dppa3 and Uhrf1 loci, respectively, Dppa3 and Uhrf1 specific gRNAs were cloned into SpCas9-hGem-T2A-Puromycin/gRNA vector, which is a modified version of SpCas9-T2A-Puromycin/gRNA vector (px459;(Ran et al., 2013), Addgene plasmid #62988) similar to that described above. To construct the homology donors plasmids, gBlocks (IDT, Coralville, IA, USA) were synthesized containing either the HALO or eGFP coding sequence flanked by homology arms with ~200-400 bp homology upstream and downstream of the gRNA target sequence at the Dppa3 or Uhrf1 locus, respectively, and then cloned into the NruI site of pUC57-GentR via cut-ligation. ESCs were transfected with equimolar amounts of gRNA and homology donor vectors. Two days after transfection, cells were plated at clonal density and subjected to a transient puromycin selection (1 ug/mL) for 40 h. After 5-6 days, ESCs positive for HALO or eGFP integration were isolated via fluorescence-activated cell sorting (FACS) and plated again at clonal density in ESC media. After 4-5 days, colonies were picked and plated on Optical bottom µClear 96-well plates and re-screened for the correct expression and localization of eGFP or HALO using live-cell spinning-disk confocal imaging. Cells were subsequently genotyped using the aforementioned cell lysis strategy and further validated by Sanger sequencing(Mulholland et al., 2015). Column purification For RRBS, genomic DNA was isolated using the QIAamp DNA Mini Kit (QIAGEN), after an overnight lysis and proteinase K treatment. RRBS library preparation was performed as described previously (Boyle et al. 2012), with the following differences: bisulfite treatment was performed using the EZ DNA Methylation-Gold™ Kit (Zymo Research Corporation) according to the manufacturer's protocol. ENA-FIRST-PUBLIC 2020-09-30T04:05:08Z ENA-LAST-UPDATE 2019-10-21T13:50:45Z External Id SAMEA6104587 INSDC center name Faculty of Biology Department II Ludwig Maximilians University Munich INSDC first public 2020-09-30T04:05:08Z INSDC last update 2019-10-21T13:50:45Z INSDC status public Submitter Id E-MTAB-6785:Tet1CM_EpiLC_RRBS_rep2 broker name ArrayExpress cell line J1 cell type epiblast-like cell common name house mouse developmental stage blastocyst disease normal genotype Tet1CM organism part inner cell mass sample name E-MTAB-6785:Tet1CM_EpiLC_RRBS_rep2 scientific_name Mus musculus ERS3905269 SAMEA6104588 10090 Mus musculus Protocols: Cells were collected by trypsinization Naïve J1 mouse ESCs were cultured and differentiated into EpiLCs as described previously (Hayashi and Saitou, 2013; Mulholland et al., 2015). In brief, for both naïve ESCs and EpiLCs defined media was used, consisting of N2B27: 50% neurobasal medium (Life Technologies), 50% DMEM/F12 (Life Technologies), 2 mM L-glutamine (Life Technologies), 0.1 mM β-mercaptoethanol (Life Technologies), N2 supplement (Life Technologies), B27 serum-free supplement (Life Technologies), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma). Naïve ESCs were maintained on flasks treated with 0.2% gelatin in defined media containing 2i (1 μM PD032591 and 3 μM CHIR99021 (Axon Medchem, Netherlands)), 1000 U/mL recombinant leukemia inhibitory factor (LIF, Millipore), and 0.3% BSA (Gibco) for at least three passages before commencing differentiation. For reprogramming experiments, naive media was supplemented with freshly prepared 100 µM Vitamin C (L-ascorbic acid 2-phosphate, Sigma). For the generation of Tet1, Tet2, and Tet1/Tet2 catalytic mutants, specific gRNAs targeting the catalytic center of Tet1 and Tet2 were cloned into a modified version of the SpCas9-T2A-GFP/gRNA (px458;(Ran et al., 2013), Addgene plasmid #48138), to which we fused a truncated form of human Geminin (hGem) to SpCas9 in order to increase homology-directed repair efficiency (Gutschner et al., 2016). A 200 bp ssDNA oligonucleotide harboring the H1652Y and D1654A mutations and ~100 bp of homology to the genomic locus was synthesized (IDT, Coralville, IA, USA). For targetings in wild-type J1 ESCs, cells were transfected with a 4:1 ratio of donor oligo and Cas9/gRNA construct. Positively transfected cells were isolated based on GFP expression using fluorescence-activated cell sorting (FACS) and plated at clonal density in ESC media 2 days after transfection. Cell lysis in 96-well plates, PCR on lysates, and restriction digests were performed as previously described (Mulholland et al., 2015). Tet1 catalytic mutation was confirmed by Sanger sequencing. For insertion of the HALO or eGFP coding sequence into the endogenous Dppa3 and Uhrf1 loci, respectively, Dppa3 and Uhrf1 specific gRNAs were cloned into SpCas9-hGem-T2A-Puromycin/gRNA vector, which is a modified version of SpCas9-T2A-Puromycin/gRNA vector (px459;(Ran et al., 2013), Addgene plasmid #62988) similar to that described above. To construct the homology donors plasmids, gBlocks (IDT, Coralville, IA, USA) were synthesized containing either the HALO or eGFP coding sequence flanked by homology arms with ~200-400 bp homology upstream and downstream of the gRNA target sequence at the Dppa3 or Uhrf1 locus, respectively, and then cloned into the NruI site of pUC57-GentR via cut-ligation. ESCs were transfected with equimolar amounts of gRNA and homology donor vectors. Two days after transfection, cells were plated at clonal density and subjected to a transient puromycin selection (1 ug/mL) for 40 h. After 5-6 days, ESCs positive for HALO or eGFP integration were isolated via fluorescence-activated cell sorting (FACS) and plated again at clonal density in ESC media. After 4-5 days, colonies were picked and plated on Optical bottom µClear 96-well plates and re-screened for the correct expression and localization of eGFP or HALO using live-cell spinning-disk confocal imaging. Cells were subsequently genotyped using the aforementioned cell lysis strategy and further validated by Sanger sequencing(Mulholland et al., 2015). Column purification For RRBS, genomic DNA was isolated using the QIAamp DNA Mini Kit (QIAGEN), after an overnight lysis and proteinase K treatment. RRBS library preparation was performed as described previously (Boyle et al. 2012), with the following differences: bisulfite treatment was performed using the EZ DNA Methylation-Gold™ Kit (Zymo Research Corporation) according to the manufacturer's protocol. ENA-FIRST-PUBLIC 2020-09-30T04:05:08Z ENA-LAST-UPDATE 2019-10-21T13:50:45Z External Id SAMEA6104588 INSDC center name Faculty of Biology Department II Ludwig Maximilians University Munich INSDC first public 2020-09-30T04:05:08Z INSDC last update 2019-10-21T13:50:45Z INSDC status public Submitter Id E-MTAB-6785:Tet1CM_ESC_RRBS_rep1 broker name ArrayExpress cell line J1 cell type embryonic stem cell common name house mouse developmental stage blastocyst disease normal genotype Tet1CM organism part inner cell mass sample name E-MTAB-6785:Tet1CM_ESC_RRBS_rep1 scientific_name Mus musculus ERS3905270 SAMEA6104589 10090 Mus musculus Protocols: Cells were collected by trypsinization Naïve J1 mouse ESCs were cultured and differentiated into EpiLCs as described previously (Hayashi and Saitou, 2013; Mulholland et al., 2015). In brief, for both naïve ESCs and EpiLCs defined media was used, consisting of N2B27: 50% neurobasal medium (Life Technologies), 50% DMEM/F12 (Life Technologies), 2 mM L-glutamine (Life Technologies), 0.1 mM β-mercaptoethanol (Life Technologies), N2 supplement (Life Technologies), B27 serum-free supplement (Life Technologies), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma). Naïve ESCs were maintained on flasks treated with 0.2% gelatin in defined media containing 2i (1 μM PD032591 and 3 μM CHIR99021 (Axon Medchem, Netherlands)), 1000 U/mL recombinant leukemia inhibitory factor (LIF, Millipore), and 0.3% BSA (Gibco) for at least three passages before commencing differentiation. For reprogramming experiments, naive media was supplemented with freshly prepared 100 µM Vitamin C (L-ascorbic acid 2-phosphate, Sigma). For the generation of Tet1, Tet2, and Tet1/Tet2 catalytic mutants, specific gRNAs targeting the catalytic center of Tet1 and Tet2 were cloned into a modified version of the SpCas9-T2A-GFP/gRNA (px458;(Ran et al., 2013), Addgene plasmid #48138), to which we fused a truncated form of human Geminin (hGem) to SpCas9 in order to increase homology-directed repair efficiency (Gutschner et al., 2016). A 200 bp ssDNA oligonucleotide harboring the H1652Y and D1654A mutations and ~100 bp of homology to the genomic locus was synthesized (IDT, Coralville, IA, USA). For targetings in wild-type J1 ESCs, cells were transfected with a 4:1 ratio of donor oligo and Cas9/gRNA construct. Positively transfected cells were isolated based on GFP expression using fluorescence-activated cell sorting (FACS) and plated at clonal density in ESC media 2 days after transfection. Cell lysis in 96-well plates, PCR on lysates, and restriction digests were performed as previously described (Mulholland et al., 2015). Tet1 catalytic mutation was confirmed by Sanger sequencing. For insertion of the HALO or eGFP coding sequence into the endogenous Dppa3 and Uhrf1 loci, respectively, Dppa3 and Uhrf1 specific gRNAs were cloned into SpCas9-hGem-T2A-Puromycin/gRNA vector, which is a modified version of SpCas9-T2A-Puromycin/gRNA vector (px459;(Ran et al., 2013), Addgene plasmid #62988) similar to that described above. To construct the homology donors plasmids, gBlocks (IDT, Coralville, IA, USA) were synthesized containing either the HALO or eGFP coding sequence flanked by homology arms with ~200-400 bp homology upstream and downstream of the gRNA target sequence at the Dppa3 or Uhrf1 locus, respectively, and then cloned into the NruI site of pUC57-GentR via cut-ligation. ESCs were transfected with equimolar amounts of gRNA and homology donor vectors. Two days after transfection, cells were plated at clonal density and subjected to a transient puromycin selection (1 ug/mL) for 40 h. After 5-6 days, ESCs positive for HALO or eGFP integration were isolated via fluorescence-activated cell sorting (FACS) and plated again at clonal density in ESC media. After 4-5 days, colonies were picked and plated on Optical bottom µClear 96-well plates and re-screened for the correct expression and localization of eGFP or HALO using live-cell spinning-disk confocal imaging. Cells were subsequently genotyped using the aforementioned cell lysis strategy and further validated by Sanger sequencing(Mulholland et al., 2015). Column purification For RRBS, genomic DNA was isolated using the QIAamp DNA Mini Kit (QIAGEN), after an overnight lysis and proteinase K treatment. RRBS library preparation was performed as described previously (Boyle et al. 2012), with the following differences: bisulfite treatment was performed using the EZ DNA Methylation-Gold™ Kit (Zymo Research Corporation) according to the manufacturer's protocol. ENA-FIRST-PUBLIC 2020-09-30T04:05:08Z ENA-LAST-UPDATE 2019-10-21T13:50:45Z External Id SAMEA6104589 INSDC center name Faculty of Biology Department II Ludwig Maximilians University Munich INSDC first public 2020-09-30T04:05:08Z INSDC last update 2019-10-21T13:50:45Z INSDC status public Submitter Id E-MTAB-6785:Tet1CM_ESC_RRBS_rep2 broker name ArrayExpress cell line J1 cell type embryonic stem cell common name house mouse developmental stage blastocyst disease normal genotype Tet1CM organism part inner cell mass sample name E-MTAB-6785:Tet1CM_ESC_RRBS_rep2 scientific_name Mus musculus ERS3905271 SAMEA6104590 10090 Mus musculus Protocols: Cells were collected by trypsinization To differentiate naïve ESCs into Epiblast-like cells (EpiLCs), flasks were first pre-treated with Geltrex (Life Technologies) diluted 1:100 in DMEM/F12 (Life Technologies) and incubated at 37 °C overnight. Naïve ESCs were plated on Geltrex-treated flasks in defined medium containing 10 ng/mL Fgf2 (R&D Systems), 20 ng/mL Activin A (R&D Systems) and 0.1× Knockout Serum Replacement (KSR) (Life Technologies). Media was changed after 24 h and EpiLCs were harvested for RRBS and RNA-seq experiments after 48 h. For the generation of Tet1, Tet2, and Tet1/Tet2 catalytic mutants, specific gRNAs targeting the catalytic center of Tet1 and Tet2 were cloned into a modified version of the SpCas9-T2A-GFP/gRNA (px458;(Ran et al., 2013), Addgene plasmid #48138), to which we fused a truncated form of human Geminin (hGem) to SpCas9 in order to increase homology-directed repair efficiency (Gutschner et al., 2016). A 200 bp ssDNA oligonucleotide harboring the H1652Y and D1654A mutations and ~100 bp of homology to the genomic locus was synthesized (IDT, Coralville, IA, USA). For targetings in wild-type J1 ESCs, cells were transfected with a 4:1 ratio of donor oligo and Cas9/gRNA construct. Positively transfected cells were isolated based on GFP expression using fluorescence-activated cell sorting (FACS) and plated at clonal density in ESC media 2 days after transfection. Cell lysis in 96-well plates, PCR on lysates, and restriction digests were performed as previously described (Mulholland et al., 2015). Tet1 catalytic mutation was confirmed by Sanger sequencing. For insertion of the HALO or eGFP coding sequence into the endogenous Dppa3 and Uhrf1 loci, respectively, Dppa3 and Uhrf1 specific gRNAs were cloned into SpCas9-hGem-T2A-Puromycin/gRNA vector, which is a modified version of SpCas9-T2A-Puromycin/gRNA vector (px459;(Ran et al., 2013), Addgene plasmid #62988) similar to that described above. To construct the homology donors plasmids, gBlocks (IDT, Coralville, IA, USA) were synthesized containing either the HALO or eGFP coding sequence flanked by homology arms with ~200-400 bp homology upstream and downstream of the gRNA target sequence at the Dppa3 or Uhrf1 locus, respectively, and then cloned into the NruI site of pUC57-GentR via cut-ligation. ESCs were transfected with equimolar amounts of gRNA and homology donor vectors. Two days after transfection, cells were plated at clonal density and subjected to a transient puromycin selection (1 ug/mL) for 40 h. After 5-6 days, ESCs positive for HALO or eGFP integration were isolated via fluorescence-activated cell sorting (FACS) and plated again at clonal density in ESC media. After 4-5 days, colonies were picked and plated on Optical bottom µClear 96-well plates and re-screened for the correct expression and localization of eGFP or HALO using live-cell spinning-disk confocal imaging. Cells were subsequently genotyped using the aforementioned cell lysis strategy and further validated by Sanger sequencing(Mulholland et al., 2015). Column purification For RRBS, genomic DNA was isolated using the QIAamp DNA Mini Kit (QIAGEN), after an overnight lysis and proteinase K treatment. RRBS library preparation was performed as described previously (Boyle et al. 2012), with the following differences: bisulfite treatment was performed using the EZ DNA Methylation-Gold™ Kit (Zymo Research Corporation) according to the manufacturer's protocol. ENA-FIRST-PUBLIC 2020-09-30T04:05:08Z ENA-LAST-UPDATE 2019-10-21T13:50:45Z External Id SAMEA6104590 INSDC center name Faculty of Biology Department II Ludwig Maximilians University Munich INSDC first public 2020-09-30T04:05:08Z INSDC last update 2019-10-21T13:50:45Z INSDC status public Submitter Id E-MTAB-6785:Tet2CM_EpiLC_RRBS_rep1 broker name ArrayExpress cell line J1 cell type epiblast-like cell common name house mouse developmental stage blastocyst disease normal genotype Tet2CM organism part inner cell mass sample name E-MTAB-6785:Tet2CM_EpiLC_RRBS_rep1 scientific_name Mus musculus ERS3905272 SAMEA6104591 10090 Mus musculus Protocols: Cells were collected by trypsinization To differentiate naïve ESCs into Epiblast-like cells (EpiLCs), flasks were first pre-treated with Geltrex (Life Technologies) diluted 1:100 in DMEM/F12 (Life Technologies) and incubated at 37 °C overnight. Naïve ESCs were plated on Geltrex-treated flasks in defined medium containing 10 ng/mL Fgf2 (R&D Systems), 20 ng/mL Activin A (R&D Systems) and 0.1× Knockout Serum Replacement (KSR) (Life Technologies). Media was changed after 24 h and EpiLCs were harvested for RRBS and RNA-seq experiments after 48 h. For the generation of Tet1, Tet2, and Tet1/Tet2 catalytic mutants, specific gRNAs targeting the catalytic center of Tet1 and Tet2 were cloned into a modified version of the SpCas9-T2A-GFP/gRNA (px458;(Ran et al., 2013), Addgene plasmid #48138), to which we fused a truncated form of human Geminin (hGem) to SpCas9 in order to increase homology-directed repair efficiency (Gutschner et al., 2016). A 200 bp ssDNA oligonucleotide harboring the H1652Y and D1654A mutations and ~100 bp of homology to the genomic locus was synthesized (IDT, Coralville, IA, USA). For targetings in wild-type J1 ESCs, cells were transfected with a 4:1 ratio of donor oligo and Cas9/gRNA construct. Positively transfected cells were isolated based on GFP expression using fluorescence-activated cell sorting (FACS) and plated at clonal density in ESC media 2 days after transfection. Cell lysis in 96-well plates, PCR on lysates, and restriction digests were performed as previously described (Mulholland et al., 2015). Tet1 catalytic mutation was confirmed by Sanger sequencing. For insertion of the HALO or eGFP coding sequence into the endogenous Dppa3 and Uhrf1 loci, respectively, Dppa3 and Uhrf1 specific gRNAs were cloned into SpCas9-hGem-T2A-Puromycin/gRNA vector, which is a modified version of SpCas9-T2A-Puromycin/gRNA vector (px459;(Ran et al., 2013), Addgene plasmid #62988) similar to that described above. To construct the homology donors plasmids, gBlocks (IDT, Coralville, IA, USA) were synthesized containing either the HALO or eGFP coding sequence flanked by homology arms with ~200-400 bp homology upstream and downstream of the gRNA target sequence at the Dppa3 or Uhrf1 locus, respectively, and then cloned into the NruI site of pUC57-GentR via cut-ligation. ESCs were transfected with equimolar amounts of gRNA and homology donor vectors. Two days after transfection, cells were plated at clonal density and subjected to a transient puromycin selection (1 ug/mL) for 40 h. After 5-6 days, ESCs positive for HALO or eGFP integration were isolated via fluorescence-activated cell sorting (FACS) and plated again at clonal density in ESC media. After 4-5 days, colonies were picked and plated on Optical bottom µClear 96-well plates and re-screened for the correct expression and localization of eGFP or HALO using live-cell spinning-disk confocal imaging. Cells were subsequently genotyped using the aforementioned cell lysis strategy and further validated by Sanger sequencing(Mulholland et al., 2015). Column purification For RRBS, genomic DNA was isolated using the QIAamp DNA Mini Kit (QIAGEN), after an overnight lysis and proteinase K treatment. RRBS library preparation was performed as described previously (Boyle et al. 2012), with the following differences: bisulfite treatment was performed using the EZ DNA Methylation-Gold™ Kit (Zymo Research Corporation) according to the manufacturer's protocol. ENA-FIRST-PUBLIC 2020-09-30T04:05:08Z ENA-LAST-UPDATE 2019-10-21T13:50:45Z External Id SAMEA6104591 INSDC center name Faculty of Biology Department II Ludwig Maximilians University Munich INSDC first public 2020-09-30T04:05:08Z INSDC last update 2019-10-21T13:50:45Z INSDC status public Submitter Id E-MTAB-6785:Tet2CM_EpiLC_RRBS_rep2 broker name ArrayExpress cell line J1 cell type epiblast-like cell common name house mouse developmental stage blastocyst disease normal genotype Tet2CM organism part inner cell mass sample name E-MTAB-6785:Tet2CM_EpiLC_RRBS_rep2 scientific_name Mus musculus ERS3905273 SAMEA6104592 10090 Mus musculus Protocols: Cells were collected by trypsinization Naïve J1 mouse ESCs were cultured and differentiated into EpiLCs as described previously (Hayashi and Saitou, 2013; Mulholland et al., 2015). In brief, for both naïve ESCs and EpiLCs defined media was used, consisting of N2B27: 50% neurobasal medium (Life Technologies), 50% DMEM/F12 (Life Technologies), 2 mM L-glutamine (Life Technologies), 0.1 mM β-mercaptoethanol (Life Technologies), N2 supplement (Life Technologies), B27 serum-free supplement (Life Technologies), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma). Naïve ESCs were maintained on flasks treated with 0.2% gelatin in defined media containing 2i (1 μM PD032591 and 3 μM CHIR99021 (Axon Medchem, Netherlands)), 1000 U/mL recombinant leukemia inhibitory factor (LIF, Millipore), and 0.3% BSA (Gibco) for at least three passages before commencing differentiation. For reprogramming experiments, naive media was supplemented with freshly prepared 100 µM Vitamin C (L-ascorbic acid 2-phosphate, Sigma). For the generation of Tet1, Tet2, and Tet1/Tet2 catalytic mutants, specific gRNAs targeting the catalytic center of Tet1 and Tet2 were cloned into a modified version of the SpCas9-T2A-GFP/gRNA (px458;(Ran et al., 2013), Addgene plasmid #48138), to which we fused a truncated form of human Geminin (hGem) to SpCas9 in order to increase homology-directed repair efficiency (Gutschner et al., 2016). A 200 bp ssDNA oligonucleotide harboring the H1652Y and D1654A mutations and ~100 bp of homology to the genomic locus was synthesized (IDT, Coralville, IA, USA). For targetings in wild-type J1 ESCs, cells were transfected with a 4:1 ratio of donor oligo and Cas9/gRNA construct. Positively transfected cells were isolated based on GFP expression using fluorescence-activated cell sorting (FACS) and plated at clonal density in ESC media 2 days after transfection. Cell lysis in 96-well plates, PCR on lysates, and restriction digests were performed as previously described (Mulholland et al., 2015). Tet1 catalytic mutation was confirmed by Sanger sequencing. For insertion of the HALO or eGFP coding sequence into the endogenous Dppa3 and Uhrf1 loci, respectively, Dppa3 and Uhrf1 specific gRNAs were cloned into SpCas9-hGem-T2A-Puromycin/gRNA vector, which is a modified version of SpCas9-T2A-Puromycin/gRNA vector (px459;(Ran et al., 2013), Addgene plasmid #62988) similar to that described above. To construct the homology donors plasmids, gBlocks (IDT, Coralville, IA, USA) were synthesized containing either the HALO or eGFP coding sequence flanked by homology arms with ~200-400 bp homology upstream and downstream of the gRNA target sequence at the Dppa3 or Uhrf1 locus, respectively, and then cloned into the NruI site of pUC57-GentR via cut-ligation. ESCs were transfected with equimolar amounts of gRNA and homology donor vectors. Two days after transfection, cells were plated at clonal density and subjected to a transient puromycin selection (1 ug/mL) for 40 h. After 5-6 days, ESCs positive for HALO or eGFP integration were isolated via fluorescence-activated cell sorting (FACS) and plated again at clonal density in ESC media. After 4-5 days, colonies were picked and plated on Optical bottom µClear 96-well plates and re-screened for the correct expression and localization of eGFP or HALO using live-cell spinning-disk confocal imaging. Cells were subsequently genotyped using the aforementioned cell lysis strategy and further validated by Sanger sequencing(Mulholland et al., 2015). Column purification For RRBS, genomic DNA was isolated using the QIAamp DNA Mini Kit (QIAGEN), after an overnight lysis and proteinase K treatment. RRBS library preparation was performed as described previously (Boyle et al. 2012), with the following differences: bisulfite treatment was performed using the EZ DNA Methylation-Gold™ Kit (Zymo Research Corporation) according to the manufacturer's protocol. ENA-FIRST-PUBLIC 2020-09-30T04:05:08Z ENA-LAST-UPDATE 2019-10-21T13:50:46Z External Id SAMEA6104592 INSDC center name Faculty of Biology Department II Ludwig Maximilians University Munich INSDC first public 2020-09-30T04:05:08Z INSDC last update 2019-10-21T13:50:46Z INSDC status public Submitter Id E-MTAB-6785:Tet2CM_ESC_RRBS_rep1 broker name ArrayExpress cell line J1 cell type embryonic stem cell common name house mouse developmental stage blastocyst disease normal genotype Tet2CM organism part inner cell mass sample name E-MTAB-6785:Tet2CM_ESC_RRBS_rep1 scientific_name Mus musculus ERS3905274 SAMEA6104593 10090 Mus musculus Protocols: Cells were collected by trypsinization Naïve J1 mouse ESCs were cultured and differentiated into EpiLCs as described previously (Hayashi and Saitou, 2013; Mulholland et al., 2015). In brief, for both naïve ESCs and EpiLCs defined media was used, consisting of N2B27: 50% neurobasal medium (Life Technologies), 50% DMEM/F12 (Life Technologies), 2 mM L-glutamine (Life Technologies), 0.1 mM β-mercaptoethanol (Life Technologies), N2 supplement (Life Technologies), B27 serum-free supplement (Life Technologies), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma). Naïve ESCs were maintained on flasks treated with 0.2% gelatin in defined media containing 2i (1 μM PD032591 and 3 μM CHIR99021 (Axon Medchem, Netherlands)), 1000 U/mL recombinant leukemia inhibitory factor (LIF, Millipore), and 0.3% BSA (Gibco) for at least three passages before commencing differentiation. For reprogramming experiments, naive media was supplemented with freshly prepared 100 µM Vitamin C (L-ascorbic acid 2-phosphate, Sigma). For the generation of Tet1, Tet2, and Tet1/Tet2 catalytic mutants, specific gRNAs targeting the catalytic center of Tet1 and Tet2 were cloned into a modified version of the SpCas9-T2A-GFP/gRNA (px458;(Ran et al., 2013), Addgene plasmid #48138), to which we fused a truncated form of human Geminin (hGem) to SpCas9 in order to increase homology-directed repair efficiency (Gutschner et al., 2016). A 200 bp ssDNA oligonucleotide harboring the H1652Y and D1654A mutations and ~100 bp of homology to the genomic locus was synthesized (IDT, Coralville, IA, USA). For targetings in wild-type J1 ESCs, cells were transfected with a 4:1 ratio of donor oligo and Cas9/gRNA construct. Positively transfected cells were isolated based on GFP expression using fluorescence-activated cell sorting (FACS) and plated at clonal density in ESC media 2 days after transfection. Cell lysis in 96-well plates, PCR on lysates, and restriction digests were performed as previously described (Mulholland et al., 2015). Tet1 catalytic mutation was confirmed by Sanger sequencing. For insertion of the HALO or eGFP coding sequence into the endogenous Dppa3 and Uhrf1 loci, respectively, Dppa3 and Uhrf1 specific gRNAs were cloned into SpCas9-hGem-T2A-Puromycin/gRNA vector, which is a modified version of SpCas9-T2A-Puromycin/gRNA vector (px459;(Ran et al., 2013), Addgene plasmid #62988) similar to that described above. To construct the homology donors plasmids, gBlocks (IDT, Coralville, IA, USA) were synthesized containing either the HALO or eGFP coding sequence flanked by homology arms with ~200-400 bp homology upstream and downstream of the gRNA target sequence at the Dppa3 or Uhrf1 locus, respectively, and then cloned into the NruI site of pUC57-GentR via cut-ligation. ESCs were transfected with equimolar amounts of gRNA and homology donor vectors. Two days after transfection, cells were plated at clonal density and subjected to a transient puromycin selection (1 ug/mL) for 40 h. After 5-6 days, ESCs positive for HALO or eGFP integration were isolated via fluorescence-activated cell sorting (FACS) and plated again at clonal density in ESC media. After 4-5 days, colonies were picked and plated on Optical bottom µClear 96-well plates and re-screened for the correct expression and localization of eGFP or HALO using live-cell spinning-disk confocal imaging. Cells were subsequently genotyped using the aforementioned cell lysis strategy and further validated by Sanger sequencing(Mulholland et al., 2015). Column purification For RRBS, genomic DNA was isolated using the QIAamp DNA Mini Kit (QIAGEN), after an overnight lysis and proteinase K treatment. RRBS library preparation was performed as described previously (Boyle et al. 2012), with the following differences: bisulfite treatment was performed using the EZ DNA Methylation-Gold™ Kit (Zymo Research Corporation) according to the manufacturer's protocol. ENA-FIRST-PUBLIC 2020-09-30T04:05:08Z ENA-LAST-UPDATE 2019-10-21T13:50:46Z External Id SAMEA6104593 INSDC center name Faculty of Biology Department II Ludwig Maximilians University Munich INSDC first public 2020-09-30T04:05:08Z INSDC last update 2019-10-21T13:50:46Z INSDC status public Submitter Id E-MTAB-6785:Tet2CM_ESC_RRBS_rep2 broker name ArrayExpress cell line J1 cell type embryonic stem cell common name house mouse developmental stage blastocyst disease normal genotype Tet2CM organism part inner cell mass sample name E-MTAB-6785:Tet2CM_ESC_RRBS_rep2 scientific_name Mus musculus ERS3905275 SAMEA6104594 10090 Mus musculus Protocols: Cells were collected by trypsinization To differentiate naïve ESCs into Epiblast-like cells (EpiLCs), flasks were first pre-treated with Geltrex (Life Technologies) diluted 1:100 in DMEM/F12 (Life Technologies) and incubated at 37 °C overnight. Naïve ESCs were plated on Geltrex-treated flasks in defined medium containing 10 ng/mL Fgf2 (R&D Systems), 20 ng/mL Activin A (R&D Systems) and 0.1× Knockout Serum Replacement (KSR) (Life Technologies). Media was changed after 24 h and EpiLCs were harvested for RRBS and RNA-seq experiments after 48 h. For the generation of Tet1, Tet2, and Tet1/Tet2 catalytic mutants, specific gRNAs targeting the catalytic center of Tet1 and Tet2 were cloned into a modified version of the SpCas9-T2A-GFP/gRNA (px458;(Ran et al., 2013), Addgene plasmid #48138), to which we fused a truncated form of human Geminin (hGem) to SpCas9 in order to increase homology-directed repair efficiency (Gutschner et al., 2016). A 200 bp ssDNA oligonucleotide harboring the H1652Y and D1654A mutations and ~100 bp of homology to the genomic locus was synthesized (IDT, Coralville, IA, USA). For targetings in wild-type J1 ESCs, cells were transfected with a 4:1 ratio of donor oligo and Cas9/gRNA construct. Positively transfected cells were isolated based on GFP expression using fluorescence-activated cell sorting (FACS) and plated at clonal density in ESC media 2 days after transfection. Cell lysis in 96-well plates, PCR on lysates, and restriction digests were performed as previously described (Mulholland et al., 2015). Tet1 catalytic mutation was confirmed by Sanger sequencing. For insertion of the HALO or eGFP coding sequence into the endogenous Dppa3 and Uhrf1 loci, respectively, Dppa3 and Uhrf1 specific gRNAs were cloned into SpCas9-hGem-T2A-Puromycin/gRNA vector, which is a modified version of SpCas9-T2A-Puromycin/gRNA vector (px459;(Ran et al., 2013), Addgene plasmid #62988) similar to that described above. To construct the homology donors plasmids, gBlocks (IDT, Coralville, IA, USA) were synthesized containing either the HALO or eGFP coding sequence flanked by homology arms with ~200-400 bp homology upstream and downstream of the gRNA target sequence at the Dppa3 or Uhrf1 locus, respectively, and then cloned into the NruI site of pUC57-GentR via cut-ligation. ESCs were transfected with equimolar amounts of gRNA and homology donor vectors. Two days after transfection, cells were plated at clonal density and subjected to a transient puromycin selection (1 ug/mL) for 40 h. After 5-6 days, ESCs positive for HALO or eGFP integration were isolated via fluorescence-activated cell sorting (FACS) and plated again at clonal density in ESC media. After 4-5 days, colonies were picked and plated on Optical bottom µClear 96-well plates and re-screened for the correct expression and localization of eGFP or HALO using live-cell spinning-disk confocal imaging. Cells were subsequently genotyped using the aforementioned cell lysis strategy and further validated by Sanger sequencing(Mulholland et al., 2015). Column purification For RRBS, genomic DNA was isolated using the QIAamp DNA Mini Kit (QIAGEN), after an overnight lysis and proteinase K treatment. RRBS library preparation was performed as described previously (Boyle et al. 2012), with the following differences: bisulfite treatment was performed using the EZ DNA Methylation-Gold™ Kit (Zymo Research Corporation) according to the manufacturer's protocol. ENA-FIRST-PUBLIC 2020-09-30T04:05:08Z ENA-LAST-UPDATE 2019-10-21T13:50:46Z External Id SAMEA6104594 INSDC center name Faculty of Biology Department II Ludwig Maximilians University Munich INSDC first public 2020-09-30T04:05:08Z INSDC last update 2019-10-21T13:50:46Z INSDC status public Submitter Id E-MTAB-6785:Tet2Tet2CM_EpiLC_RRBS_rep1 broker name ArrayExpress cell line J1 cell type epiblast-like cell common name house mouse developmental stage blastocyst disease normal genotype Tet1Tet2CM organism part inner cell mass sample name E-MTAB-6785:Tet2Tet2CM_EpiLC_RRBS_rep1 scientific_name Mus musculus ERS3905276 SAMEA6104595 10090 Mus musculus Protocols: Cells were collected by trypsinization To differentiate naïve ESCs into Epiblast-like cells (EpiLCs), flasks were first pre-treated with Geltrex (Life Technologies) diluted 1:100 in DMEM/F12 (Life Technologies) and incubated at 37 °C overnight. Naïve ESCs were plated on Geltrex-treated flasks in defined medium containing 10 ng/mL Fgf2 (R&D Systems), 20 ng/mL Activin A (R&D Systems) and 0.1× Knockout Serum Replacement (KSR) (Life Technologies). Media was changed after 24 h and EpiLCs were harvested for RRBS and RNA-seq experiments after 48 h. For the generation of Tet1, Tet2, and Tet1/Tet2 catalytic mutants, specific gRNAs targeting the catalytic center of Tet1 and Tet2 were cloned into a modified version of the SpCas9-T2A-GFP/gRNA (px458;(Ran et al., 2013), Addgene plasmid #48138), to which we fused a truncated form of human Geminin (hGem) to SpCas9 in order to increase homology-directed repair efficiency (Gutschner et al., 2016). A 200 bp ssDNA oligonucleotide harboring the H1652Y and D1654A mutations and ~100 bp of homology to the genomic locus was synthesized (IDT, Coralville, IA, USA). For targetings in wild-type J1 ESCs, cells were transfected with a 4:1 ratio of donor oligo and Cas9/gRNA construct. Positively transfected cells were isolated based on GFP expression using fluorescence-activated cell sorting (FACS) and plated at clonal density in ESC media 2 days after transfection. Cell lysis in 96-well plates, PCR on lysates, and restriction digests were performed as previously described (Mulholland et al., 2015). Tet1 catalytic mutation was confirmed by Sanger sequencing. For insertion of the HALO or eGFP coding sequence into the endogenous Dppa3 and Uhrf1 loci, respectively, Dppa3 and Uhrf1 specific gRNAs were cloned into SpCas9-hGem-T2A-Puromycin/gRNA vector, which is a modified version of SpCas9-T2A-Puromycin/gRNA vector (px459;(Ran et al., 2013), Addgene plasmid #62988) similar to that described above. To construct the homology donors plasmids, gBlocks (IDT, Coralville, IA, USA) were synthesized containing either the HALO or eGFP coding sequence flanked by homology arms with ~200-400 bp homology upstream and downstream of the gRNA target sequence at the Dppa3 or Uhrf1 locus, respectively, and then cloned into the NruI site of pUC57-GentR via cut-ligation. ESCs were transfected with equimolar amounts of gRNA and homology donor vectors. Two days after transfection, cells were plated at clonal density and subjected to a transient puromycin selection (1 ug/mL) for 40 h. After 5-6 days, ESCs positive for HALO or eGFP integration were isolated via fluorescence-activated cell sorting (FACS) and plated again at clonal density in ESC media. After 4-5 days, colonies were picked and plated on Optical bottom µClear 96-well plates and re-screened for the correct expression and localization of eGFP or HALO using live-cell spinning-disk confocal imaging. Cells were subsequently genotyped using the aforementioned cell lysis strategy and further validated by Sanger sequencing(Mulholland et al., 2015). Column purification For RRBS, genomic DNA was isolated using the QIAamp DNA Mini Kit (QIAGEN), after an overnight lysis and proteinase K treatment. RRBS library preparation was performed as described previously (Boyle et al. 2012), with the following differences: bisulfite treatment was performed using the EZ DNA Methylation-Gold™ Kit (Zymo Research Corporation) according to the manufacturer's protocol. ENA-FIRST-PUBLIC 2020-09-30T04:05:08Z ENA-LAST-UPDATE 2019-10-21T13:50:46Z External Id SAMEA6104595 INSDC center name Faculty of Biology Department II Ludwig Maximilians University Munich INSDC first public 2020-09-30T04:05:08Z INSDC last update 2019-10-21T13:50:46Z INSDC status public Submitter Id E-MTAB-6785:Tet2Tet2CM_EpiLC_RRBS_rep2 broker name ArrayExpress cell line J1 cell type epiblast-like cell common name house mouse developmental stage blastocyst disease normal genotype Tet1Tet2CM organism part inner cell mass sample name E-MTAB-6785:Tet2Tet2CM_EpiLC_RRBS_rep2 scientific_name Mus musculus ERS3905277 SAMEA6104596 10090 Mus musculus Protocols: Cells were collected by trypsinization Naïve J1 mouse ESCs were cultured and differentiated into EpiLCs as described previously (Hayashi and Saitou, 2013; Mulholland et al., 2015). In brief, for both naïve ESCs and EpiLCs defined media was used, consisting of N2B27: 50% neurobasal medium (Life Technologies), 50% DMEM/F12 (Life Technologies), 2 mM L-glutamine (Life Technologies), 0.1 mM β-mercaptoethanol (Life Technologies), N2 supplement (Life Technologies), B27 serum-free supplement (Life Technologies), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma). Naïve ESCs were maintained on flasks treated with 0.2% gelatin in defined media containing 2i (1 μM PD032591 and 3 μM CHIR99021 (Axon Medchem, Netherlands)), 1000 U/mL recombinant leukemia inhibitory factor (LIF, Millipore), and 0.3% BSA (Gibco) for at least three passages before commencing differentiation. For reprogramming experiments, naive media was supplemented with freshly prepared 100 µM Vitamin C (L-ascorbic acid 2-phosphate, Sigma). For the generation of Tet1, Tet2, and Tet1/Tet2 catalytic mutants, specific gRNAs targeting the catalytic center of Tet1 and Tet2 were cloned into a modified version of the SpCas9-T2A-GFP/gRNA (px458;(Ran et al., 2013), Addgene plasmid #48138), to which we fused a truncated form of human Geminin (hGem) to SpCas9 in order to increase homology-directed repair efficiency (Gutschner et al., 2016). A 200 bp ssDNA oligonucleotide harboring the H1652Y and D1654A mutations and ~100 bp of homology to the genomic locus was synthesized (IDT, Coralville, IA, USA). For targetings in wild-type J1 ESCs, cells were transfected with a 4:1 ratio of donor oligo and Cas9/gRNA construct. Positively transfected cells were isolated based on GFP expression using fluorescence-activated cell sorting (FACS) and plated at clonal density in ESC media 2 days after transfection. Cell lysis in 96-well plates, PCR on lysates, and restriction digests were performed as previously described (Mulholland et al., 2015). Tet1 catalytic mutation was confirmed by Sanger sequencing. For insertion of the HALO or eGFP coding sequence into the endogenous Dppa3 and Uhrf1 loci, respectively, Dppa3 and Uhrf1 specific gRNAs were cloned into SpCas9-hGem-T2A-Puromycin/gRNA vector, which is a modified version of SpCas9-T2A-Puromycin/gRNA vector (px459;(Ran et al., 2013), Addgene plasmid #62988) similar to that described above. To construct the homology donors plasmids, gBlocks (IDT, Coralville, IA, USA) were synthesized containing either the HALO or eGFP coding sequence flanked by homology arms with ~200-400 bp homology upstream and downstream of the gRNA target sequence at the Dppa3 or Uhrf1 locus, respectively, and then cloned into the NruI site of pUC57-GentR via cut-ligation. ESCs were transfected with equimolar amounts of gRNA and homology donor vectors. Two days after transfection, cells were plated at clonal density and subjected to a transient puromycin selection (1 ug/mL) for 40 h. After 5-6 days, ESCs positive for HALO or eGFP integration were isolated via fluorescence-activated cell sorting (FACS) and plated again at clonal density in ESC media. After 4-5 days, colonies were picked and plated on Optical bottom µClear 96-well plates and re-screened for the correct expression and localization of eGFP or HALO using live-cell spinning-disk confocal imaging. Cells were subsequently genotyped using the aforementioned cell lysis strategy and further validated by Sanger sequencing(Mulholland et al., 2015). Column purification For RRBS, genomic DNA was isolated using the QIAamp DNA Mini Kit (QIAGEN), after an overnight lysis and proteinase K treatment. RRBS library preparation was performed as described previously (Boyle et al. 2012), with the following differences: bisulfite treatment was performed using the EZ DNA Methylation-Gold™ Kit (Zymo Research Corporation) according to the manufacturer's protocol. ENA-FIRST-PUBLIC 2020-09-30T04:05:08Z ENA-LAST-UPDATE 2019-10-21T13:50:46Z External Id SAMEA6104596 INSDC center name Faculty of Biology Department II Ludwig Maximilians University Munich INSDC first public 2020-09-30T04:05:08Z INSDC last update 2019-10-21T13:50:46Z INSDC status public Submitter Id E-MTAB-6785:Tet2Tet2CM_ESC_RRBS_rep1 broker name ArrayExpress cell line J1 cell type embryonic stem cell common name house mouse developmental stage blastocyst disease normal genotype Tet1Tet2CM organism part inner cell mass sample name E-MTAB-6785:Tet2Tet2CM_ESC_RRBS_rep1 scientific_name Mus musculus ERS3905278 SAMEA6104597 10090 Mus musculus Protocols: Cells were collected by trypsinization Naïve J1 mouse ESCs were cultured and differentiated into EpiLCs as described previously (Hayashi and Saitou, 2013; Mulholland et al., 2015). In brief, for both naïve ESCs and EpiLCs defined media was used, consisting of N2B27: 50% neurobasal medium (Life Technologies), 50% DMEM/F12 (Life Technologies), 2 mM L-glutamine (Life Technologies), 0.1 mM β-mercaptoethanol (Life Technologies), N2 supplement (Life Technologies), B27 serum-free supplement (Life Technologies), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma). Naïve ESCs were maintained on flasks treated with 0.2% gelatin in defined media containing 2i (1 μM PD032591 and 3 μM CHIR99021 (Axon Medchem, Netherlands)), 1000 U/mL recombinant leukemia inhibitory factor (LIF, Millipore), and 0.3% BSA (Gibco) for at least three passages before commencing differentiation. For reprogramming experiments, naive media was supplemented with freshly prepared 100 µM Vitamin C (L-ascorbic acid 2-phosphate, Sigma). For the generation of Tet1, Tet2, and Tet1/Tet2 catalytic mutants, specific gRNAs targeting the catalytic center of Tet1 and Tet2 were cloned into a modified version of the SpCas9-T2A-GFP/gRNA (px458;(Ran et al., 2013), Addgene plasmid #48138), to which we fused a truncated form of human Geminin (hGem) to SpCas9 in order to increase homology-directed repair efficiency (Gutschner et al., 2016). A 200 bp ssDNA oligonucleotide harboring the H1652Y and D1654A mutations and ~100 bp of homology to the genomic locus was synthesized (IDT, Coralville, IA, USA). For targetings in wild-type J1 ESCs, cells were transfected with a 4:1 ratio of donor oligo and Cas9/gRNA construct. Positively transfected cells were isolated based on GFP expression using fluorescence-activated cell sorting (FACS) and plated at clonal density in ESC media 2 days after transfection. Cell lysis in 96-well plates, PCR on lysates, and restriction digests were performed as previously described (Mulholland et al., 2015). Tet1 catalytic mutation was confirmed by Sanger sequencing. For insertion of the HALO or eGFP coding sequence into the endogenous Dppa3 and Uhrf1 loci, respectively, Dppa3 and Uhrf1 specific gRNAs were cloned into SpCas9-hGem-T2A-Puromycin/gRNA vector, which is a modified version of SpCas9-T2A-Puromycin/gRNA vector (px459;(Ran et al., 2013), Addgene plasmid #62988) similar to that described above. To construct the homology donors plasmids, gBlocks (IDT, Coralville, IA, USA) were synthesized containing either the HALO or eGFP coding sequence flanked by homology arms with ~200-400 bp homology upstream and downstream of the gRNA target sequence at the Dppa3 or Uhrf1 locus, respectively, and then cloned into the NruI site of pUC57-GentR via cut-ligation. ESCs were transfected with equimolar amounts of gRNA and homology donor vectors. Two days after transfection, cells were plated at clonal density and subjected to a transient puromycin selection (1 ug/mL) for 40 h. After 5-6 days, ESCs positive for HALO or eGFP integration were isolated via fluorescence-activated cell sorting (FACS) and plated again at clonal density in ESC media. After 4-5 days, colonies were picked and plated on Optical bottom µClear 96-well plates and re-screened for the correct expression and localization of eGFP or HALO using live-cell spinning-disk confocal imaging. Cells were subsequently genotyped using the aforementioned cell lysis strategy and further validated by Sanger sequencing(Mulholland et al., 2015). Column purification For RRBS, genomic DNA was isolated using the QIAamp DNA Mini Kit (QIAGEN), after an overnight lysis and proteinase K treatment. RRBS library preparation was performed as described previously (Boyle et al. 2012), with the following differences: bisulfite treatment was performed using the EZ DNA Methylation-Gold™ Kit (Zymo Research Corporation) according to the manufacturer's protocol. ENA-FIRST-PUBLIC 2020-09-30T04:05:08Z ENA-LAST-UPDATE 2019-10-21T13:50:46Z External Id SAMEA6104597 INSDC center name Faculty of Biology Department II Ludwig Maximilians University Munich INSDC first public 2020-09-30T04:05:08Z INSDC last update 2019-10-21T13:50:46Z INSDC status public Submitter Id E-MTAB-6785:Tet2Tet2CM_ESC_RRBS_rep2 broker name ArrayExpress cell line J1 cell type embryonic stem cell common name house mouse developmental stage blastocyst disease normal genotype Tet1Tet2CM organism part inner cell mass sample name E-MTAB-6785:Tet2Tet2CM_ESC_RRBS_rep2 scientific_name Mus musculus ERS3905279 SAMEA6104598 10090 Mus musculus Protocols: Cells were collected by trypsinization To differentiate naïve ESCs into Epiblast-like cells (EpiLCs), flasks were first pre-treated with Geltrex (Life Technologies) diluted 1:100 in DMEM/F12 (Life Technologies) and incubated at 37 °C overnight. Naïve ESCs were plated on Geltrex-treated flasks in defined medium containing 10 ng/mL Fgf2 (R&D Systems), 20 ng/mL Activin A (R&D Systems) and 0.1× Knockout Serum Replacement (KSR) (Life Technologies). Media was changed after 24 h and EpiLCs were harvested for RRBS and RNA-seq experiments after 48 h. Column purification For RRBS, genomic DNA was isolated using the QIAamp DNA Mini Kit (QIAGEN), after an overnight lysis and proteinase K treatment. RRBS library preparation was performed as described previously (Boyle et al. 2012), with the following differences: bisulfite treatment was performed using the EZ DNA Methylation-Gold™ Kit (Zymo Research Corporation) according to the manufacturer's protocol. ENA-FIRST-PUBLIC 2020-09-30T04:05:08Z ENA-LAST-UPDATE 2019-10-21T13:50:46Z External Id SAMEA6104598 INSDC center name Faculty of Biology Department II Ludwig Maximilians University Munich INSDC first public 2020-09-30T04:05:08Z INSDC last update 2019-10-21T13:50:46Z INSDC status public Submitter Id E-MTAB-6785:wt_EpiLC_RRBS_rep1 broker name ArrayExpress cell line J1 cell type epiblast-like cell common name house mouse developmental stage blastocyst disease normal genotype wild type genotype organism part inner cell mass sample name E-MTAB-6785:wt_EpiLC_RRBS_rep1 scientific_name Mus musculus ERS3905280 SAMEA6104599 10090 Mus musculus Protocols: Cells were collected by trypsinization To differentiate naïve ESCs into Epiblast-like cells (EpiLCs), flasks were first pre-treated with Geltrex (Life Technologies) diluted 1:100 in DMEM/F12 (Life Technologies) and incubated at 37 °C overnight. Naïve ESCs were plated on Geltrex-treated flasks in defined medium containing 10 ng/mL Fgf2 (R&D Systems), 20 ng/mL Activin A (R&D Systems) and 0.1× Knockout Serum Replacement (KSR) (Life Technologies). Media was changed after 24 h and EpiLCs were harvested for RRBS and RNA-seq experiments after 48 h. Column purification For RRBS, genomic DNA was isolated using the QIAamp DNA Mini Kit (QIAGEN), after an overnight lysis and proteinase K treatment. RRBS library preparation was performed as described previously (Boyle et al. 2012), with the following differences: bisulfite treatment was performed using the EZ DNA Methylation-Gold™ Kit (Zymo Research Corporation) according to the manufacturer's protocol. ENA-FIRST-PUBLIC 2020-09-30T04:05:08Z ENA-LAST-UPDATE 2019-10-21T13:50:46Z External Id SAMEA6104599 INSDC center name Faculty of Biology Department II Ludwig Maximilians University Munich INSDC first public 2020-09-30T04:05:08Z INSDC last update 2019-10-21T13:50:46Z INSDC status public Submitter Id E-MTAB-6785:wt_EpiLC_RRBS_rep2 broker name ArrayExpress cell line J1 cell type epiblast-like cell common name house mouse developmental stage blastocyst disease normal genotype wild type genotype organism part inner cell mass sample name E-MTAB-6785:wt_EpiLC_RRBS_rep2 scientific_name Mus musculus ERS3905281 SAMEA6104600 10090 Mus musculus Protocols: Cells were collected by trypsinization Naïve J1 mouse ESCs were cultured and differentiated into EpiLCs as described previously (Hayashi and Saitou, 2013; Mulholland et al., 2015). In brief, for both naïve ESCs and EpiLCs defined media was used, consisting of N2B27: 50% neurobasal medium (Life Technologies), 50% DMEM/F12 (Life Technologies), 2 mM L-glutamine (Life Technologies), 0.1 mM β-mercaptoethanol (Life Technologies), N2 supplement (Life Technologies), B27 serum-free supplement (Life Technologies), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma). Naïve ESCs were maintained on flasks treated with 0.2% gelatin in defined media containing 2i (1 μM PD032591 and 3 μM CHIR99021 (Axon Medchem, Netherlands)), 1000 U/mL recombinant leukemia inhibitory factor (LIF, Millipore), and 0.3% BSA (Gibco) for at least three passages before commencing differentiation. For reprogramming experiments, naive media was supplemented with freshly prepared 100 µM Vitamin C (L-ascorbic acid 2-phosphate, Sigma). Column purification For RRBS, genomic DNA was isolated using the QIAamp DNA Mini Kit (QIAGEN), after an overnight lysis and proteinase K treatment. RRBS library preparation was performed as described previously (Boyle et al. 2012), with the following differences: bisulfite treatment was performed using the EZ DNA Methylation-Gold™ Kit (Zymo Research Corporation) according to the manufacturer's protocol. ENA-FIRST-PUBLIC 2020-09-30T04:05:08Z ENA-LAST-UPDATE 2019-10-21T13:50:46Z External Id SAMEA6104600 INSDC center name Faculty of Biology Department II Ludwig Maximilians University Munich INSDC first public 2020-09-30T04:05:08Z INSDC last update 2019-10-21T13:50:46Z INSDC status public Submitter Id E-MTAB-6785:wt_ESC_RRBS_rep1 broker name ArrayExpress cell line J1 cell type embryonic stem cell common name house mouse developmental stage blastocyst disease normal genotype wild type genotype organism part inner cell mass sample name E-MTAB-6785:wt_ESC_RRBS_rep1 scientific_name Mus musculus ERS3905282 SAMEA6104601 10090 Mus musculus Protocols: Cells were collected by trypsinization Naïve J1 mouse ESCs were cultured and differentiated into EpiLCs as described previously (Hayashi and Saitou, 2013; Mulholland et al., 2015). In brief, for both naïve ESCs and EpiLCs defined media was used, consisting of N2B27: 50% neurobasal medium (Life Technologies), 50% DMEM/F12 (Life Technologies), 2 mM L-glutamine (Life Technologies), 0.1 mM β-mercaptoethanol (Life Technologies), N2 supplement (Life Technologies), B27 serum-free supplement (Life Technologies), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma). Naïve ESCs were maintained on flasks treated with 0.2% gelatin in defined media containing 2i (1 μM PD032591 and 3 μM CHIR99021 (Axon Medchem, Netherlands)), 1000 U/mL recombinant leukemia inhibitory factor (LIF, Millipore), and 0.3% BSA (Gibco) for at least three passages before commencing differentiation. For reprogramming experiments, naive media was supplemented with freshly prepared 100 µM Vitamin C (L-ascorbic acid 2-phosphate, Sigma). Column purification For RRBS, genomic DNA was isolated using the QIAamp DNA Mini Kit (QIAGEN), after an overnight lysis and proteinase K treatment. RRBS library preparation was performed as described previously (Boyle et al. 2012), with the following differences: bisulfite treatment was performed using the EZ DNA Methylation-Gold™ Kit (Zymo Research Corporation) according to the manufacturer's protocol. ENA-FIRST-PUBLIC 2020-09-30T04:05:08Z ENA-LAST-UPDATE 2019-10-21T13:50:46Z External Id SAMEA6104601 INSDC center name Faculty of Biology Department II Ludwig Maximilians University Munich INSDC first public 2020-09-30T04:05:08Z INSDC last update 2019-10-21T13:50:46Z INSDC status public Submitter Id E-MTAB-6785:wt_ESC_RRBS_rep2 broker name ArrayExpress cell line J1 cell type embryonic stem cell common name house mouse developmental stage blastocyst disease normal genotype wild type genotype organism part inner cell mass sample name E-MTAB-6785:wt_ESC_RRBS_rep2 scientific_name Mus musculus