mii oocytes were collected from superovulated 45-week - old c57bl/6babr mice , under a stereomicroscope , by mouth pipetting , and stored at 80c . prior to scbs - seq , 2 oocyte lysis buffer ( 10 mm tris - cl ph7.4 , 2% sds ) and 0.5l proteinase k were added ( final volume 12l ) followed by incubation at 37c for 1h . e14 escs were cultured in serum plus lif or 2i plus lif conditions as described previously . the 2i escs had been maintained in this medium for 24 days and matched serum escs were cultured in parallel . single escs were collected by facs in 12l of esc lysis buffer ( 10 mm tris - cl ph7.4 , 0.6% sds , 0.5l proteinase k ) using a bd influx instrument in single cell 1 drop mode . topro-3 and hoechst 33342 staining were used to select for live cells with low dna content ( i.e. in g0/g1 ) . escs were incubated at 37c for 1h and stored at 20c until required for library preparation . negative controls were either lysis buffer alone (  empty  tubes ) or sorted bd accudrop beads , and were prepared and processed concomitantly with all single cell samples . bisulfite conversion was performed on cell lysates using the imprint dna modification kit ( sigma ) with the following modifications : all volumes were halved , and chemical denaturation was followed by incubation at 65c for 90min , 95c for 3min and 65c for 20min . purification was performed as described previously , and dna eluted in 10 mm tris - cl ( ph 8.5 ) and combined with 0.4 mm dntps , 0.4m oligo1 ( [ btn]ctacacgacgctcttccgatctnnnnnnnnn ) and 1 blue buffer ( sigma ) ( 24l final ) before incubation at 65c for 3min followed by 4c pause . 50u of klenow exo- ( sigma ) were added and the samples incubated at 4c for 5min , + 1c/15s to 37c , 37c for 30min . samples were incubated at 95c for 1min and transferred immediately to ice before addition of fresh oligo1 ( 10pmol ) , klenow exo- ( 25u ) , and dntps ( 1nmol ) in 2.5l total . the samples were incubated at 4c for 5min , + 1c/15s to 37c , 37c for 30min . this random priming and extension was repeated a further 3 times ( 5 rounds in total ) . i ( neb ) for 1h at 37c before dna was purified using 0.8 agencourt ampure xp beads ( beckman coulter ) according to the manufacturer s guidelines . samples were eluted in 10 mm tris - cl ( ph 8.5 ) and incubated with washed m-280 streptavidin dynabeads ( life technologies ) for 20min with rotation at room temperature . beads were washed twice with 0.1n naoh , and twice with 10 mm tris - cl ( ph 8.5 ) and re - suspended in 48l of 0.4 mm dntps , 0.4m oligo2 ( tgctgaaccgctcttccgatctnnnnnnnnn ) and 1 blue buffer . samples were incubated at 95c for 45s and transferred immediately to ice before addition of 100u klenow exo- ( sigma ) and incubation at 4c for 5min , + 1c/15s to 37c , 37c for 90min . beads were washed with 10 mm tris - cl ( ph 8.5 ) and resuspended in 50l of 0.4 mm dntps , 0.4m pe1.0 forward primer ( aatgatacggcgaccaccgagatctacactctttc - cctacacgacgctcttccgatct ) , 0.4m indexed ipcrtag reverse primer , 1u kapa hifi hotstart dna polymerase ( kapa biosystems ) in 1 hifi fidelity buffer . libraries were then amplified by pcr as follows : 95c 2min , 12 - 13 repeats of ( 94c 80s , 65c 30s , 72c 30s ) , 72c 3min , 4c hold . amplified libraries were purified using 0.8 agencourt ampure xp beads , according to the manufacturer s guidelines , and were assessed for quality and quantity using high - sensitivity dna chips on the agilent bioanalyser , and the kapa library quantification kit for illumina ( kapa biosystems ) . pools of 12 - 14 single cell libraries were prepared for 100bp paired - end sequencing on a hiseq2500 in rapid - run mode ( 2 lanes / run ) . samples from bulk cell populations were prepared according to the protocol above , with some modifications . for the bulk oocyte sample , 120 mii oocytes were collected and lysed as described above . for esc bulk cell samples , dna was purified from cell pellets using the qiaamp micro kit ( qiagen ) , according to the manufacturer s instructions , and 50ng of purified dna was used in the library preparation . one round of first strand synthesis was performed using 0.8 mm dntps and 4m oligo1 , and second strand synthesis also used 0.8 mm dntps and 4m oligo2 . raw sequence reads were trimmed to remove the first 9 base pairs , adapter contamination and poor quality reads using trim galore ( v0.3.5 , www.bioinformatics.babraham.ac.uk/projects/trim_galore/ , parameters : --clip_r1 9 --clip_r2 9 --paired ) . due to the multiple rounds of random priming performed with oligo1 , scbs - seq libraries are non - directional . trimmed sequences were first mapped to the human genome ( build grch37 ) using bismark ( v0.10.1 ; parameters : --pe , --bowtie2 , --non_directional , -- unmapped ) , resulting in 1.4% mapping efficiency ( 0.2 - 13.2% range ) . remaining sequences were mapped to the mouse genome ( build ncbi37 ) in single - end mode ( bismark parameters : --bowtie2 -- non_directional ) . methylation calls were extracted after duplicate sequences had been excluded . for oocyte bulk analysis , our mii bulk dataset was merged in silico with previously published datasets8 ( ddbj / genbank / embl accession number dra000570 ) . data visualization and analysis were performed using seqmonk , custom r and java scripts . for figure 1c , cg methylation was calculated as the average of methylation for each cpg position , and non - cpg methylation was extracted from the bismark reports . percentage of concordance was calculated as the percentage of cpgs presenting the same methylation call at the same genomic position across two cells . for correlation analysis ( pearson s ) , 2 kb windows were defined informative if at least 8 cpgs per window were sequenced . hyper - methylated and hypo - methylated cgis were defined as 80% and 20% methylation respectively . 12 and supplementary table 2 ) were extracted from previously published datasets . statistical analysis for estimating sample - specific methylation rates , estimating mean methylation rates and for clustering mii oocytes were collected from superovulated 45-week - old c57bl/6babr mice , under a stereomicroscope , by mouth pipetting , and stored at 80c . prior to scbs - seq , 2 oocyte lysis buffer ( 10 mm tris - cl ph7.4 , 2% sds ) and 0.5l proteinase k were added ( final volume 12l ) followed by incubation at 37c for 1h . e14 escs were cultured in serum plus lif or 2i plus lif conditions as described previously . the 2i escs had been maintained in this medium for 24 days and matched serum escs were cultured in parallel . single escs were collected by facs in 12l of esc lysis buffer ( 10 mm tris - cl ph7.4 , 0.6% sds , 0.5l proteinase k ) using a bd influx instrument in single cell 1 drop mode . topro-3 and hoechst 33342 staining were used to select for live cells with low dna content ( i.e. in g0/g1 ) . escs were incubated at 37c for 1h and stored at 20c until required for library preparation . negative controls were either lysis buffer alone (  empty  tubes ) or sorted bd accudrop beads , and were prepared and processed concomitantly with all single cell samples . bisulfite conversion was performed on cell lysates using the imprint dna modification kit ( sigma ) with the following modifications : all volumes were halved , and chemical denaturation was followed by incubation at 65c for 90min , 95c for 3min and 65c for 20min . purification was performed as described previously , and dna eluted in 10 mm tris - cl ( ph 8.5 ) and combined with 0.4 mm dntps , 0.4m oligo1 ( [ btn]ctacacgacgctcttccgatctnnnnnnnnn ) and 1 blue buffer ( sigma ) ( 24l final ) before incubation at 65c for 3min followed by 4c pause . 50u of klenow exo- ( sigma ) were added and the samples incubated at 4c for 5min , + 1c/15s to 37c , 37c for 30min . samples were incubated at 95c for 1min and transferred immediately to ice before addition of fresh oligo1 ( 10pmol ) , klenow exo- ( 25u ) , and dntps ( 1nmol ) in 2.5l total . the samples were incubated at 4c for 5min , + 1c/15s to 37c , 37c for 30min . this random priming and extension was repeated a further 3 times ( 5 rounds in total ) . samples were then incubated with 40u exonuclease i ( neb ) for 1h at 37c before dna was purified using 0.8 agencourt ampure xp beads ( beckman coulter ) according to the manufacturer s guidelines . samples were eluted in 10 mm tris - cl ( ph 8.5 ) and incubated with washed m-280 streptavidin dynabeads ( life technologies ) for 20min with rotation at room temperature . beads were washed twice with 0.1n naoh , and twice with 10 mm tris - cl ( ph 8.5 ) and re - suspended in 48l of 0.4 mm dntps , 0.4m oligo2 ( tgctgaaccgctcttccgatctnnnnnnnnn ) and 1 blue buffer . samples were incubated at 95c for 45s and transferred immediately to ice before addition of 100u klenow exo- ( sigma ) and incubation at 4c for 5min , + 1c/15s to 37c , 37c for 90min . beads were washed with 10 mm tris - cl ( ph 8.5 ) and resuspended in 50l of 0.4 mm dntps , 0.4m pe1.0 forward primer ( aatgatacggcgaccaccgagatctacactctttc - cctacacgacgctcttccgatct ) , 0.4m indexed ipcrtag reverse primer , 1u kapa hifi hotstart dna polymerase ( kapa biosystems ) in 1 hifi fidelity buffer . libraries were then amplified by pcr as follows : 95c 2min , 12 - 13 repeats of ( 94c 80s , 65c 30s , 72c 30s ) , 72c 3min , 4c hold . amplified libraries were purified using 0.8 agencourt ampure xp beads , according to the manufacturer s guidelines , and were assessed for quality and quantity using high - sensitivity dna chips on the agilent bioanalyser , and the kapa library quantification kit for illumina ( kapa biosystems ) . pools of 12 - 14 single cell libraries were prepared for 100bp paired - end sequencing on a hiseq2500 in rapid - run mode ( 2 lanes / run ) . samples from bulk cell populations were prepared according to the protocol above , with some modifications . for the bulk oocyte sample , 120 mii oocytes were collected and lysed as described above . for esc bulk cell samples , dna was purified from cell pellets using the qiaamp micro kit ( qiagen ) , according to the manufacturer s instructions , and 50ng of purified dna was used in the library preparation . one round of first strand synthesis was performed using 0.8 mm dntps and 4m oligo1 , and second strand synthesis also used 0.8 mm dntps and 4m oligo2 . raw sequence reads were trimmed to remove the first 9 base pairs , adapter contamination and poor quality reads using trim galore ( v0.3.5 , www.bioinformatics.babraham.ac.uk/projects/trim_galore/ , parameters : --clip_r1 9 --clip_r2 9 --paired ) . due to the multiple rounds of random priming performed with oligo1 , trimmed sequences were first mapped to the human genome ( build grch37 ) using bismark ( v0.10.1 ; parameters : --pe , --bowtie2 , --non_directional , -- unmapped ) , resulting in 1.4% mapping efficiency ( 0.2 - 13.2% range ) . remaining sequences were mapped to the mouse genome ( build ncbi37 ) in single - end mode ( bismark parameters : --bowtie2 -- non_directional ) . bulk analysis , our mii bulk dataset was merged in silico with previously published datasets8 ( ddbj / genbank / embl accession number dra000570 ) . data visualization and analysis were performed using seqmonk , custom r and java scripts . for figure 1c , cg methylation was calculated as the average of methylation for each cpg position , and non - cpg methylation was extracted from the bismark reports . percentage of concordance was calculated as the percentage of cpgs presenting the same methylation call at the same genomic position across two cells . for correlation analysis ( pearson s ) , 2 kb windows were defined informative if at least 8 cpgs per window were sequenced . hyper - methylated and hypo - methylated cgis were defined as 80% and 20% methylation respectively . statistical analysis for estimating sample - specific methylation rates , estimating mean methylation rates and for clustering are detailed in supplementary note 1 .
we report a single - cell bisulfite sequencing method ( scbs - seq ) capable of accurately measuring dna methylation at up to 48.4% of cpgs . we observed that escs grown in serum or 2i both display epigenetic heterogeneity , with 
 2i - like  cells present in serum cultures . in silico integration of 12 individual mouse oocyte datasets 
 largely recapitulates the whole dna methylome , making scbs - seq a versatile tool to explore dna methylation in rare cells and heterogeneous populations .