SRX5171448 GSM3525807 SRP174096 SRS4179070 GSM3525807 Bisulfite-Seq GENOMIC RANDOM Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 6.5 (E6.5). Natural timed matings were set up between Dnmt3a floxed/floxed, Dnmt3b floxed/floxed, Zp3 +ve B6/129 females (resulting in ablation of DNA methylation in the oocyte) and CAST males (denoted as DKO.CAST). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. E6.5 epiblast (N=4) and ExE (N=8) samples were pooled (an estimated ~2500 cells), washed in PBS, and then flash frozen in 10µL of nuclear lysis buffer (Sigma). Samples were thawed on ice and permeabilised with 0.1% Triton-X/0.1% deoxycholate in PBS on ice. MNase digestion was completed using 200U of micrococcal nuclease (New England Biolabs) in prepared digestion buffer, as previously described (Brind'Amour et al. 2015 Nat Comm). Samples were digested at 21°C for 7.5 minutes and the reaction was stopped with 100mM EDTA. Prior to the immunoprecipitation, one 10% input was taken from each chromatin sample for bisulphite sequencing. DNA from immunoprecipitated chromatin or input was purified using solid phase reversible immobilization (SPRI) purification using Sera-Mag carboxylate-modified Magnetic SpeedBeads (Fisher Scientific) at a 1.8:1 ratio. DNA samples were then bisulfite converted using the EZ DNA Methylation Direct kit (Zymo Research). The resulting DNA was purified using columns from EZ DNA Methylation Direct kit (Zymo Research). First strand synthesis was then performed using Klenow Exo- (New England Biolabs) with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This was followed by exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (ThermoFisher Scientific). Samples were then subjected to second strand synthesis similar to the first strand synthesis, followed by 12 PCR amplification cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 75bp paired-end sequencing on Illumina NextSeq500. NextSeq 500 gds 303525807 GSM3525807 GEO Accession GSM3525807 SRX5171449 GSM3525808 SRP174096 SRS4179071 GSM3525808 Bisulfite-Seq GENOMIC RANDOM Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 6.5 (E6.5). Natural timed matings were set up between Dnmt3a floxed/floxed, Dnmt3b floxed/floxed, Zp3 +ve B6/129 females (resulting in ablation of DNA methylation in the oocyte) and CAST males (denoted as DKO.CAST). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. E6.5 epiblast (N=4) and ExE (N=8) samples were pooled (an estimated ~2500 cells), washed in PBS, and then flash frozen in 10µL of nuclear lysis buffer (Sigma). Samples were thawed on ice and permeabilised with 0.1% Triton-X/0.1% deoxycholate in PBS on ice. MNase digestion was completed using 200U of micrococcal nuclease (New England Biolabs) in prepared digestion buffer, as previously described (Brind'Amour et al. 2015 Nat Comm). Samples were digested at 21°C for 7.5 minutes and the reaction was stopped with 100mM EDTA. Prior to the immunoprecipitation, one 10% input was taken from each chromatin sample for bisulphite sequencing. DNA from immunoprecipitated chromatin or input was purified using solid phase reversible immobilization (SPRI) purification using Sera-Mag carboxylate-modified Magnetic SpeedBeads (Fisher Scientific) at a 1.8:1 ratio. DNA samples were then bisulfite converted using the EZ DNA Methylation Direct kit (Zymo Research). The resulting DNA was purified using columns from EZ DNA Methylation Direct kit (Zymo Research). First strand synthesis was then performed using Klenow Exo- (New England Biolabs) with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This was followed by exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (ThermoFisher Scientific). Samples were then subjected to second strand synthesis similar to the first strand synthesis, followed by 12 PCR amplification cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 75bp paired-end sequencing on Illumina NextSeq500. NextSeq 500 gds 303525808 GSM3525808 GEO Accession GSM3525808 SRX5171450 GSM3525809 SRP174096 SRS4179072 GSM3525809 Bisulfite-Seq GENOMIC RANDOM Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 6.5 (E6.5). Natural timed matings were set up between Dnmt3a floxed/floxed, Dnmt3b floxed/floxed, Zp3 +ve B6/129 females (resulting in ablation of DNA methylation in the oocyte) and CAST males (denoted as DKO.CAST). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. E6.5 epiblast (N=4) and ExE (N=8) samples were pooled (an estimated ~2500 cells), washed in PBS, and then flash frozen in 10µL of nuclear lysis buffer (Sigma). Samples were thawed on ice and permeabilised with 0.1% Triton-X/0.1% deoxycholate in PBS on ice. MNase digestion was completed using 200U of micrococcal nuclease (New England Biolabs) in prepared digestion buffer, as previously described (Brind'Amour et al. 2015 Nat Comm). Samples were digested at 21°C for 7.5 minutes and the reaction was stopped with 100mM EDTA. Prior to the immunoprecipitation, one 10% input was taken from each chromatin sample for bisulphite sequencing. DNA from immunoprecipitated chromatin or input was purified using solid phase reversible immobilization (SPRI) purification using Sera-Mag carboxylate-modified Magnetic SpeedBeads (Fisher Scientific) at a 1.8:1 ratio. DNA samples were then bisulfite converted using the EZ DNA Methylation Direct kit (Zymo Research). The resulting DNA was purified using columns from EZ DNA Methylation Direct kit (Zymo Research). First strand synthesis was then performed using Klenow Exo- (New England Biolabs) with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This was followed by exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (ThermoFisher Scientific). Samples were then subjected to second strand synthesis similar to the first strand synthesis, followed by 12 PCR amplification cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 75bp paired-end sequencing on Illumina NextSeq500. NextSeq 500 gds 303525809 GSM3525809 GEO Accession GSM3525809 SRX5171451 GSM3525810 SRP174096 SRS4179073 GSM3525810 Bisulfite-Seq GENOMIC RANDOM Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 6.5 (E6.5). Natural timed matings were set up between Dnmt3a floxed/floxed, Dnmt3b floxed/floxed, Zp3 +ve B6/129 females (resulting in ablation of DNA methylation in the oocyte) and CAST males (denoted as DKO.CAST). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. E6.5 epiblast (N=4) and ExE (N=8) samples were pooled (an estimated ~2500 cells), washed in PBS, and then flash frozen in 10µL of nuclear lysis buffer (Sigma). Samples were thawed on ice and permeabilised with 0.1% Triton-X/0.1% deoxycholate in PBS on ice. MNase digestion was completed using 200U of micrococcal nuclease (New England Biolabs) in prepared digestion buffer, as previously described (Brind'Amour et al. 2015 Nat Comm). Samples were digested at 21°C for 7.5 minutes and the reaction was stopped with 100mM EDTA. Prior to the immunoprecipitation, one 10% input was taken from each chromatin sample for bisulphite sequencing. DNA from immunoprecipitated chromatin or input was purified using solid phase reversible immobilization (SPRI) purification using Sera-Mag carboxylate-modified Magnetic SpeedBeads (Fisher Scientific) at a 1.8:1 ratio. DNA samples were then bisulfite converted using the EZ DNA Methylation Direct kit (Zymo Research). The resulting DNA was purified using columns from EZ DNA Methylation Direct kit (Zymo Research). First strand synthesis was then performed using Klenow Exo- (New England Biolabs) with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This was followed by exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (ThermoFisher Scientific). Samples were then subjected to second strand synthesis similar to the first strand synthesis, followed by 12 PCR amplification cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 75bp paired-end sequencing on Illumina NextSeq500. NextSeq 500 gds 303525810 GSM3525810 GEO Accession GSM3525810 SRX5171452 GSM3525811 SRP174096 SRS4179074 GSM3525811 Bisulfite-Seq GENOMIC RANDOM Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 6.5 (E6.5). Natural timed matings were set up between Dnmt3a floxed/floxed, Dnmt3b floxed/floxed, Zp3 +ve B6/129 females (resulting in ablation of DNA methylation in the oocyte) and CAST males (denoted as DKO.CAST). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. E6.5 epiblast (N=4) and ExE (N=8) samples were pooled (an estimated ~2500 cells), washed in PBS, and then flash frozen in 10µL of nuclear lysis buffer (Sigma). Samples were thawed on ice and permeabilised with 0.1% Triton-X/0.1% deoxycholate in PBS on ice. MNase digestion was completed using 200U of micrococcal nuclease (New England Biolabs) in prepared digestion buffer, as previously described (Brind'Amour et al. 2015 Nat Comm). Samples were digested at 21°C for 7.5 minutes and the reaction was stopped with 100mM EDTA. Prior to the immunoprecipitation, one 10% input was taken from each chromatin sample for bisulphite sequencing. DNA from immunoprecipitated chromatin or input was purified using solid phase reversible immobilization (SPRI) purification using Sera-Mag carboxylate-modified Magnetic SpeedBeads (Fisher Scientific) at a 1.8:1 ratio. DNA samples were then bisulfite converted using the EZ DNA Methylation Direct kit (Zymo Research). The resulting DNA was purified using columns from EZ DNA Methylation Direct kit (Zymo Research). First strand synthesis was then performed using Klenow Exo- (New England Biolabs) with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This was followed by exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (ThermoFisher Scientific). Samples were then subjected to second strand synthesis similar to the first strand synthesis, followed by 12 PCR amplification cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 75bp paired-end sequencing on Illumina NextSeq500. NextSeq 500 gds 303525811 GSM3525811 GEO Accession GSM3525811 SRX5171453 GSM3525812 SRP174096 SRS4179076 GSM3525812 Bisulfite-Seq GENOMIC RANDOM Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 6.5 (E6.5). Natural timed matings were set up between Dnmt3a floxed/floxed, Dnmt3b floxed/floxed, Zp3 +ve B6/129 females (resulting in ablation of DNA methylation in the oocyte) and CAST males (denoted as DKO.CAST). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. E6.5 epiblast (N=4) and ExE (N=8) samples were pooled (an estimated ~2500 cells), washed in PBS, and then flash frozen in 10µL of nuclear lysis buffer (Sigma). Samples were thawed on ice and permeabilised with 0.1% Triton-X/0.1% deoxycholate in PBS on ice. MNase digestion was completed using 200U of micrococcal nuclease (New England Biolabs) in prepared digestion buffer, as previously described (Brind'Amour et al. 2015 Nat Comm). Samples were digested at 21°C for 7.5 minutes and the reaction was stopped with 100mM EDTA. Prior to the immunoprecipitation, one 10% input was taken from each chromatin sample for bisulphite sequencing. DNA from immunoprecipitated chromatin or input was purified using solid phase reversible immobilization (SPRI) purification using Sera-Mag carboxylate-modified Magnetic SpeedBeads (Fisher Scientific) at a 1.8:1 ratio. DNA samples were then bisulfite converted using the EZ DNA Methylation Direct kit (Zymo Research). The resulting DNA was purified using columns from EZ DNA Methylation Direct kit (Zymo Research). First strand synthesis was then performed using Klenow Exo- (New England Biolabs) with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This was followed by exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (ThermoFisher Scientific). Samples were then subjected to second strand synthesis similar to the first strand synthesis, followed by 12 PCR amplification cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 75bp paired-end sequencing on Illumina NextSeq500. NextSeq 500 gds 303525812 GSM3525812 GEO Accession GSM3525812 SRX5171454 GSM3525813 SRP174096 SRS4179075 GSM3525813 Bisulfite-Seq GENOMIC RANDOM Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 6.5 (E6.5). Natural timed matings were set up between Dnmt3a floxed/floxed, Dnmt3b floxed/floxed, Zp3 +ve B6/129 females (resulting in ablation of DNA methylation in the oocyte) and CAST males (denoted as DKO.CAST). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. E6.5 epiblast (N=4) and ExE (N=8) samples were pooled (an estimated ~2500 cells), washed in PBS, and then flash frozen in 10µL of nuclear lysis buffer (Sigma). Samples were thawed on ice and permeabilised with 0.1% Triton-X/0.1% deoxycholate in PBS on ice. MNase digestion was completed using 200U of micrococcal nuclease (New England Biolabs) in prepared digestion buffer, as previously described (Brind'Amour et al. 2015 Nat Comm). Samples were digested at 21°C for 7.5 minutes and the reaction was stopped with 100mM EDTA. Prior to the immunoprecipitation, one 10% input was taken from each chromatin sample for bisulphite sequencing. DNA from immunoprecipitated chromatin or input was purified using solid phase reversible immobilization (SPRI) purification using Sera-Mag carboxylate-modified Magnetic SpeedBeads (Fisher Scientific) at a 1.8:1 ratio. DNA samples were then bisulfite converted using the EZ DNA Methylation Direct kit (Zymo Research). The resulting DNA was purified using columns from EZ DNA Methylation Direct kit (Zymo Research). First strand synthesis was then performed using Klenow Exo- (New England Biolabs) with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This was followed by exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (ThermoFisher Scientific). Samples were then subjected to second strand synthesis similar to the first strand synthesis, followed by 12 PCR amplification cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 75bp paired-end sequencing on Illumina NextSeq500. NextSeq 500 gds 303525813 GSM3525813 GEO Accession GSM3525813 SRX5171455 GSM3525814 SRP174096 SRS4179077 GSM3525814 Bisulfite-Seq GENOMIC RANDOM Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 6.5 (E6.5). Natural timed matings were set up between Dnmt3a floxed/floxed, Dnmt3b floxed/floxed, Zp3 +ve B6/129 females (resulting in ablation of DNA methylation in the oocyte) and CAST males (denoted as DKO.CAST). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. E6.5 epiblast (N=4) and ExE (N=8) samples were pooled (an estimated ~2500 cells), washed in PBS, and then flash frozen in 10µL of nuclear lysis buffer (Sigma). Samples were thawed on ice and permeabilised with 0.1% Triton-X/0.1% deoxycholate in PBS on ice. MNase digestion was completed using 200U of micrococcal nuclease (New England Biolabs) in prepared digestion buffer, as previously described (Brind'Amour et al. 2015 Nat Comm). Samples were digested at 21°C for 7.5 minutes and the reaction was stopped with 100mM EDTA. Prior to the immunoprecipitation, one 10% input was taken from each chromatin sample for bisulphite sequencing. DNA from immunoprecipitated chromatin or input was purified using solid phase reversible immobilization (SPRI) purification using Sera-Mag carboxylate-modified Magnetic SpeedBeads (Fisher Scientific) at a 1.8:1 ratio. DNA samples were then bisulfite converted using the EZ DNA Methylation Direct kit (Zymo Research). The resulting DNA was purified using columns from EZ DNA Methylation Direct kit (Zymo Research). First strand synthesis was then performed using Klenow Exo- (New England Biolabs) with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This was followed by exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (ThermoFisher Scientific). Samples were then subjected to second strand synthesis similar to the first strand synthesis, followed by 12 PCR amplification cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 75bp paired-end sequencing on Illumina NextSeq500. NextSeq 500 gds 303525814 GSM3525814 GEO Accession GSM3525814 SRX5171456 GSM3525815 SRP174096 SRS4179078 GSM3525815 Bisulfite-Seq GENOMIC RANDOM Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 6.5 (E6.5). Natural timed matings were set up between Dnmt3a floxed/floxed, Dnmt3b floxed/floxed, Zp3 +ve B6/129 females (resulting in ablation of DNA methylation in the oocyte) and CAST males (denoted as DKO.CAST). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. E6.5 epiblast (N=4) and ExE (N=8) samples were pooled (an estimated ~2500 cells), washed in PBS, and then flash frozen in 10µL of nuclear lysis buffer (Sigma). Samples were thawed on ice and permeabilised with 0.1% Triton-X/0.1% deoxycholate in PBS on ice. MNase digestion was completed using 200U of micrococcal nuclease (New England Biolabs) in prepared digestion buffer, as previously described (Brind'Amour et al. 2015 Nat Comm). Samples were digested at 21°C for 7.5 minutes and the reaction was stopped with 100mM EDTA. Prior to the immunoprecipitation, one 10% input was taken from each chromatin sample for bisulphite sequencing. DNA from immunoprecipitated chromatin or input was purified using solid phase reversible immobilization (SPRI) purification using Sera-Mag carboxylate-modified Magnetic SpeedBeads (Fisher Scientific) at a 1.8:1 ratio. DNA samples were then bisulfite converted using the EZ DNA Methylation Direct kit (Zymo Research). The resulting DNA was purified using columns from EZ DNA Methylation Direct kit (Zymo Research). First strand synthesis was then performed using Klenow Exo- (New England Biolabs) with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This was followed by exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (ThermoFisher Scientific). Samples were then subjected to second strand synthesis similar to the first strand synthesis, followed by 12 PCR amplification cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 75bp paired-end sequencing on Illumina NextSeq500. NextSeq 500 gds 303525815 GSM3525815 GEO Accession GSM3525815 SRX5171457 GSM3525816 SRP174096 SRS4179080 GSM3525816 Bisulfite-Seq GENOMIC RANDOM Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 6.5 (E6.5). Natural timed matings were set up between Dnmt3a floxed/floxed, Dnmt3b floxed/floxed, Zp3 +ve B6/129 females (resulting in ablation of DNA methylation in the oocyte) and CAST males (denoted as DKO.CAST). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. E6.5 epiblast (N=4) and ExE (N=8) samples were pooled (an estimated ~2500 cells), washed in PBS, and then flash frozen in 10µL of nuclear lysis buffer (Sigma). Samples were thawed on ice and permeabilised with 0.1% Triton-X/0.1% deoxycholate in PBS on ice. MNase digestion was completed using 200U of micrococcal nuclease (New England Biolabs) in prepared digestion buffer, as previously described (Brind'Amour et al. 2015 Nat Comm). Samples were digested at 21°C for 7.5 minutes and the reaction was stopped with 100mM EDTA. Prior to the immunoprecipitation, one 10% input was taken from each chromatin sample for bisulphite sequencing. DNA from immunoprecipitated chromatin or input was purified using solid phase reversible immobilization (SPRI) purification using Sera-Mag carboxylate-modified Magnetic SpeedBeads (Fisher Scientific) at a 1.8:1 ratio. DNA samples were then bisulfite converted using the EZ DNA Methylation Direct kit (Zymo Research). The resulting DNA was purified using columns from EZ DNA Methylation Direct kit (Zymo Research). First strand synthesis was then performed using Klenow Exo- (New England Biolabs) with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This was followed by exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (ThermoFisher Scientific). Samples were then subjected to second strand synthesis similar to the first strand synthesis, followed by 12 PCR amplification cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 75bp paired-end sequencing on Illumina NextSeq500. NextSeq 500 gds 303525816 GSM3525816 GEO Accession GSM3525816 SRX5171458 GSM3525817 SRP174096 SRS4179079 GSM3525817 Bisulfite-Seq GENOMIC RANDOM Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 6.5 (E6.5). Natural timed matings were set up between Dnmt3a floxed/floxed, Dnmt3b floxed/floxed, Zp3 +ve B6/129 females (resulting in ablation of DNA methylation in the oocyte) and CAST males (denoted as DKO.CAST). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. E6.5 epiblast (N=4) and ExE (N=8) samples were pooled (an estimated ~2500 cells), washed in PBS, and then flash frozen in 10µL of nuclear lysis buffer (Sigma). Samples were thawed on ice and permeabilised with 0.1% Triton-X/0.1% deoxycholate in PBS on ice. MNase digestion was completed using 200U of micrococcal nuclease (New England Biolabs) in prepared digestion buffer, as previously described (Brind'Amour et al. 2015 Nat Comm). Samples were digested at 21°C for 7.5 minutes and the reaction was stopped with 100mM EDTA. Prior to the immunoprecipitation, one 10% input was taken from each chromatin sample for bisulphite sequencing. DNA from immunoprecipitated chromatin or input was purified using solid phase reversible immobilization (SPRI) purification using Sera-Mag carboxylate-modified Magnetic SpeedBeads (Fisher Scientific) at a 1.8:1 ratio. DNA samples were then bisulfite converted using the EZ DNA Methylation Direct kit (Zymo Research). The resulting DNA was purified using columns from EZ DNA Methylation Direct kit (Zymo Research). First strand synthesis was then performed using Klenow Exo- (New England Biolabs) with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This was followed by exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (ThermoFisher Scientific). Samples were then subjected to second strand synthesis similar to the first strand synthesis, followed by 12 PCR amplification cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 75bp paired-end sequencing on Illumina NextSeq500. NextSeq 500 gds 303525817 GSM3525817 GEO Accession GSM3525817 SRX5171459 GSM3525818 SRP174096 SRS4179081 GSM3525818 Bisulfite-Seq GENOMIC RANDOM Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 6.5 (E6.5). Natural timed matings were set up between Dnmt3a floxed/floxed, Dnmt3b floxed/floxed, Zp3 +ve B6/129 females (resulting in ablation of DNA methylation in the oocyte) and CAST males (denoted as DKO.CAST). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. E6.5 epiblast (N=4) and ExE (N=8) samples were pooled (an estimated ~2500 cells), washed in PBS, and then flash frozen in 10µL of nuclear lysis buffer (Sigma). Samples were thawed on ice and permeabilised with 0.1% Triton-X/0.1% deoxycholate in PBS on ice. MNase digestion was completed using 200U of micrococcal nuclease (New England Biolabs) in prepared digestion buffer, as previously described (Brind'Amour et al. 2015 Nat Comm). Samples were digested at 21°C for 7.5 minutes and the reaction was stopped with 100mM EDTA. Prior to the immunoprecipitation, one 10% input was taken from each chromatin sample for bisulphite sequencing. DNA from immunoprecipitated chromatin or input was purified using solid phase reversible immobilization (SPRI) purification using Sera-Mag carboxylate-modified Magnetic SpeedBeads (Fisher Scientific) at a 1.8:1 ratio. DNA samples were then bisulfite converted using the EZ DNA Methylation Direct kit (Zymo Research). The resulting DNA was purified using columns from EZ DNA Methylation Direct kit (Zymo Research). First strand synthesis was then performed using Klenow Exo- (New England Biolabs) with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This was followed by exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (ThermoFisher Scientific). Samples were then subjected to second strand synthesis similar to the first strand synthesis, followed by 12 PCR amplification cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 75bp paired-end sequencing on Illumina NextSeq500. NextSeq 500 gds 303525818 GSM3525818 GEO Accession GSM3525818 SRX5171460 GSM3525819 SRP174096 SRS4179082 GSM3525819 Bisulfite-Seq GENOMIC RANDOM Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 6.5 (E6.5). Natural timed matings were set up between Dnmt3a floxed/floxed, Dnmt3b floxed/floxed, Zp3 +ve B6/129 females (resulting in ablation of DNA methylation in the oocyte) and CAST males (denoted as DKO.CAST). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. E6.5 epiblast (N=4) and ExE (N=8) samples were pooled (an estimated ~2500 cells), washed in PBS, and then flash frozen in 10µL of nuclear lysis buffer (Sigma). Samples were thawed on ice and permeabilised with 0.1% Triton-X/0.1% deoxycholate in PBS on ice. MNase digestion was completed using 200U of micrococcal nuclease (New England Biolabs) in prepared digestion buffer, as previously described (Brind'Amour et al. 2015 Nat Comm). Samples were digested at 21°C for 7.5 minutes and the reaction was stopped with 100mM EDTA. Prior to the immunoprecipitation, one 10% input was taken from each chromatin sample for bisulphite sequencing. DNA from immunoprecipitated chromatin or input was purified using solid phase reversible immobilization (SPRI) purification using Sera-Mag carboxylate-modified Magnetic SpeedBeads (Fisher Scientific) at a 1.8:1 ratio. DNA samples were then bisulfite converted using the EZ DNA Methylation Direct kit (Zymo Research). The resulting DNA was purified using columns from EZ DNA Methylation Direct kit (Zymo Research). First strand synthesis was then performed using Klenow Exo- (New England Biolabs) with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This was followed by exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (ThermoFisher Scientific). Samples were then subjected to second strand synthesis similar to the first strand synthesis, followed by 12 PCR amplification cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 75bp paired-end sequencing on Illumina NextSeq500. NextSeq 500 gds 303525819 GSM3525819 GEO Accession GSM3525819 SRX5171461 GSM3525820 SRP174096 SRS4179084 GSM3525820 Bisulfite-Seq GENOMIC RANDOM Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 6.5 (E6.5). Natural timed matings were set up between Dnmt3a floxed/floxed, Dnmt3b floxed/floxed, Zp3 +ve B6/129 females (resulting in ablation of DNA methylation in the oocyte) and CAST males (denoted as DKO.CAST). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. E6.5 epiblast (N=4) and ExE (N=8) samples were pooled (an estimated ~2500 cells), washed in PBS, and then flash frozen in 10µL of nuclear lysis buffer (Sigma). Samples were thawed on ice and permeabilised with 0.1% Triton-X/0.1% deoxycholate in PBS on ice. MNase digestion was completed using 200U of micrococcal nuclease (New England Biolabs) in prepared digestion buffer, as previously described (Brind'Amour et al. 2015 Nat Comm). Samples were digested at 21°C for 7.5 minutes and the reaction was stopped with 100mM EDTA. Prior to the immunoprecipitation, one 10% input was taken from each chromatin sample for bisulphite sequencing. DNA from immunoprecipitated chromatin or input was purified using solid phase reversible immobilization (SPRI) purification using Sera-Mag carboxylate-modified Magnetic SpeedBeads (Fisher Scientific) at a 1.8:1 ratio. DNA samples were then bisulfite converted using the EZ DNA Methylation Direct kit (Zymo Research). The resulting DNA was purified using columns from EZ DNA Methylation Direct kit (Zymo Research). First strand synthesis was then performed using Klenow Exo- (New England Biolabs) with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This was followed by exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (ThermoFisher Scientific). Samples were then subjected to second strand synthesis similar to the first strand synthesis, followed by 12 PCR amplification cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 75bp paired-end sequencing on Illumina NextSeq500. NextSeq 500 gds 303525820 GSM3525820 GEO Accession GSM3525820 SRX5171462 GSM3525821 SRP174096 SRS4179083 GSM3525821 Bisulfite-Seq GENOMIC RANDOM Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 6.5 (E6.5). Natural timed matings were set up between Dnmt3a floxed/floxed, Dnmt3b floxed/floxed, Zp3 +ve B6/129 females (resulting in ablation of DNA methylation in the oocyte) and CAST males (denoted as DKO.CAST). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. E6.5 epiblast (N=4) and ExE (N=8) samples were pooled (an estimated ~2500 cells), washed in PBS, and then flash frozen in 10µL of nuclear lysis buffer (Sigma). Samples were thawed on ice and permeabilised with 0.1% Triton-X/0.1% deoxycholate in PBS on ice. MNase digestion was completed using 200U of micrococcal nuclease (New England Biolabs) in prepared digestion buffer, as previously described (Brind'Amour et al. 2015 Nat Comm). Samples were digested at 21°C for 7.5 minutes and the reaction was stopped with 100mM EDTA. Prior to the immunoprecipitation, one 10% input was taken from each chromatin sample for bisulphite sequencing. DNA from immunoprecipitated chromatin or input was purified using solid phase reversible immobilization (SPRI) purification using Sera-Mag carboxylate-modified Magnetic SpeedBeads (Fisher Scientific) at a 1.8:1 ratio. DNA samples were then bisulfite converted using the EZ DNA Methylation Direct kit (Zymo Research). The resulting DNA was purified using columns from EZ DNA Methylation Direct kit (Zymo Research). First strand synthesis was then performed using Klenow Exo- (New England Biolabs) with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This was followed by exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (ThermoFisher Scientific). Samples were then subjected to second strand synthesis similar to the first strand synthesis, followed by 12 PCR amplification cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 75bp paired-end sequencing on Illumina NextSeq500. NextSeq 500 gds 303525821 GSM3525821 GEO Accession GSM3525821 SRX6867935 GSM4084100 SRP174096 PRJNA510998 SRS5405368 GSM4084100 Bisulfite-Seq GENOMIC RANDOM Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 7.5 (E7.5). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. Individual E7.5 epiblast and ExE samples were washed in PBS, and then flash frozen in 10µL of RLT+ buffer (Qiagen). Samples were digested at 37°C for 1 hour. DNA samples were then bisulfite converted using the EZ DNA Methylation Direct kit (Zymo Research). The resulting DNA was purified using columns from EZ DNA Methylation Direct kit (Zymo Research). The bisulfite conversion reaction was repeated due to poor initial conversion. First strand synthesis was then performed using Klenow Exo- (New England Biolabs) with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This was followed by exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (ThermoFisher Scientific). Samples were then subjected to second strand synthesis similar to the first strand synthesis, followed by 10 PCR amplification cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 150bp paired-end sequencing on Illumina NextSeq500. NextSeq 500 gds 304084100 GSM4084100 GEO Accession GSM4084100 SRX6867936 GSM4084101 SRP174096 PRJNA510998 SRS5405369 GSM4084101 Bisulfite-Seq GENOMIC RANDOM Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 7.5 (E7.5). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. Individual E7.5 epiblast and ExE samples were washed in PBS, and then flash frozen in 10µL of RLT+ buffer (Qiagen). Samples were digested at 37°C for 1 hour. DNA samples were then bisulfite converted using the EZ DNA Methylation Direct kit (Zymo Research). The resulting DNA was purified using columns from EZ DNA Methylation Direct kit (Zymo Research). The bisulfite conversion reaction was repeated due to poor initial conversion. First strand synthesis was then performed using Klenow Exo- (New England Biolabs) with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This was followed by exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (ThermoFisher Scientific). Samples were then subjected to second strand synthesis similar to the first strand synthesis, followed by 10 PCR amplification cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 150bp paired-end sequencing on Illumina NextSeq500. NextSeq 500 gds 304084101 GSM4084101 GEO Accession GSM4084101 SRX6867937 GSM4084102 SRP174096 PRJNA510998 SRS5405370 GSM4084102 Bisulfite-Seq GENOMIC RANDOM Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 7.5 (E7.5). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. Individual E7.5 epiblast and ExE samples were washed in PBS, and then flash frozen in 10µL of RLT+ buffer (Qiagen). Samples were digested at 37°C for 1 hour. DNA samples were then bisulfite converted using the EZ DNA Methylation Direct kit (Zymo Research). The resulting DNA was purified using columns from EZ DNA Methylation Direct kit (Zymo Research). The bisulfite conversion reaction was repeated due to poor initial conversion. First strand synthesis was then performed using Klenow Exo- (New England Biolabs) with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This was followed by exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (ThermoFisher Scientific). Samples were then subjected to second strand synthesis similar to the first strand synthesis, followed by 10 PCR amplification cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 150bp paired-end sequencing on Illumina NextSeq500. NextSeq 500 gds 304084102 GSM4084102 GEO Accession GSM4084102 SRX6867938 GSM4084103 SRP174096 PRJNA510998 SRS5405371 GSM4084103 Bisulfite-Seq GENOMIC RANDOM Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 7.5 (E7.5). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. Individual E7.5 epiblast and ExE samples were washed in PBS, and then flash frozen in 10µL of RLT+ buffer (Qiagen). Samples were digested at 37°C for 1 hour. DNA samples were then bisulfite converted using the EZ DNA Methylation Direct kit (Zymo Research). The resulting DNA was purified using columns from EZ DNA Methylation Direct kit (Zymo Research). The bisulfite conversion reaction was repeated due to poor initial conversion. First strand synthesis was then performed using Klenow Exo- (New England Biolabs) with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This was followed by exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (ThermoFisher Scientific). Samples were then subjected to second strand synthesis similar to the first strand synthesis, followed by 10 PCR amplification cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 150bp paired-end sequencing on Illumina NextSeq500. NextSeq 500 gds 304084103 GSM4084103 GEO Accession GSM4084103 SRX6867939 GSM4084104 SRP174096 PRJNA510998 SRS5405372 GSM4084104 Bisulfite-Seq GENOMIC RANDOM Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 7.5 (E7.5). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. Individual E7.5 epiblast and ExE samples were washed in PBS, and then flash frozen in 10µL of RLT+ buffer (Qiagen). Samples were digested at 37°C for 1 hour. DNA samples were then bisulfite converted using the EZ DNA Methylation Direct kit (Zymo Research). The resulting DNA was purified using columns from EZ DNA Methylation Direct kit (Zymo Research). The bisulfite conversion reaction was repeated due to poor initial conversion. First strand synthesis was then performed using Klenow Exo- (New England Biolabs) with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This was followed by exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (ThermoFisher Scientific). Samples were then subjected to second strand synthesis similar to the first strand synthesis, followed by 10 PCR amplification cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 150bp paired-end sequencing on Illumina NextSeq500. NextSeq 500 gds 304084104 GSM4084104 GEO Accession GSM4084104 SRX6867940 GSM4084105 SRP174096 PRJNA510998 SRS5405373 GSM4084105 Bisulfite-Seq GENOMIC RANDOM Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 7.5 (E7.5). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. Individual E7.5 epiblast and ExE samples were washed in PBS, and then flash frozen in 10µL of RLT+ buffer (Qiagen). Samples were digested at 37°C for 1 hour. DNA samples were then bisulfite converted using the EZ DNA Methylation Direct kit (Zymo Research). The resulting DNA was purified using columns from EZ DNA Methylation Direct kit (Zymo Research). The bisulfite conversion reaction was repeated due to poor initial conversion. First strand synthesis was then performed using Klenow Exo- (New England Biolabs) with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This was followed by exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (ThermoFisher Scientific). Samples were then subjected to second strand synthesis similar to the first strand synthesis, followed by 10 PCR amplification cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 150bp paired-end sequencing on Illumina NextSeq500. NextSeq 500 gds 304084105 GSM4084105 GEO Accession GSM4084105 SRX6867941 GSM4084106 SRP174096 PRJNA510998 SRS5405374 GSM4084106 Bisulfite-Seq GENOMIC RANDOM Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 7.5 (E7.5). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. Individual E7.5 epiblast and ExE samples were washed in PBS, and then flash frozen in 10µL of RLT+ buffer (Qiagen). Samples were digested at 37°C for 1 hour. DNA samples were then bisulfite converted using the EZ DNA Methylation Direct kit (Zymo Research). The resulting DNA was purified using columns from EZ DNA Methylation Direct kit (Zymo Research). The bisulfite conversion reaction was repeated due to poor initial conversion. First strand synthesis was then performed using Klenow Exo- (New England Biolabs) with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This was followed by exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (ThermoFisher Scientific). Samples were then subjected to second strand synthesis similar to the first strand synthesis, followed by 10 PCR amplification cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 150bp paired-end sequencing on Illumina NextSeq500. NextSeq 500 gds 304084106 GSM4084106 GEO Accession GSM4084106 SRX6867942 GSM4084107 SRP174096 PRJNA510998 SRS5405375 GSM4084107 Bisulfite-Seq GENOMIC RANDOM Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 7.5 (E7.5). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. Individual E7.5 epiblast and ExE samples were washed in PBS, and then flash frozen in 10µL of RLT+ buffer (Qiagen). Samples were digested at 37°C for 1 hour. DNA samples were then bisulfite converted using the EZ DNA Methylation Direct kit (Zymo Research). The resulting DNA was purified using columns from EZ DNA Methylation Direct kit (Zymo Research). The bisulfite conversion reaction was repeated due to poor initial conversion. First strand synthesis was then performed using Klenow Exo- (New England Biolabs) with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This was followed by exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (ThermoFisher Scientific). Samples were then subjected to second strand synthesis similar to the first strand synthesis, followed by 10 PCR amplification cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 150bp paired-end sequencing on Illumina NextSeq500. NextSeq 500 gds 304084107 GSM4084107 GEO Accession GSM4084107