title: GSM6166045: Dnmt3l KO, E7.5, Epi, 3 (PBAT); Mus; Bisulfite-Seq
library_construction_protocol: Natural timed matings between heterozygous females and males were used for Dnmt3l, Dnmt3a, Dnmt3b and Dnmt3a/3b KO strains to generate KO and WT embryos. E7.5 epiblast, extra-embryonic ectoderm (ExE) and ectoplacental cone (EPC) for each embryo were manually separated. Single E7.5 epiblast and ExE samples were individually frozen in 10µL of buffer RLT Plus (Qiagen) for RNA-seq or PBAT; the corresponding EPC was used for genotyping.  Cells were lysed with 0.5% SDS at 37°C for 1 hour and bisulphite converted using the Imprint DNA Modification kit (Sigma). Bisulphite-converted DNA was purified using the EZ DNA Methylation Direct kit, as directed (Zymo Research). Using a biotin-conjugated adaptor containing standard Illumina adaptor sequences and 9 bp of random sequences (9N), first-strand synthesis was performed using Klenow Exo- enzyme (New England Biolabs). Following exonuclease (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (Thermo Fisher Scientific), second-strand synthesis was performed. Libraries were amplified and indexed using 10 PCR cycles with Phusion High-Fidelity DNA polymerase (New England Biolabs). Libraries were multiplexed for 75-bp paired-end sequencing on an Illumina NextSeq500. Lysed cells were bisulphite converted using the Imprint DNA Modification kit (Sigma). Bisulphite-converted DNA was purified using the EZ DNA Methylation Direct kit, as directed (Zymo Research). Using a biotin-conjugated adaptor containing standard Illumina adaptor sequences and 9 bp of random sequences (9N), first-strand synthesis was performed using Klenow Exo- enzyme (New England Biolabs). Following exonuclease (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (Thermo Fisher Scientific), second-strand synthesis was performed. Libraries were amplified and indexed using 10 PCR cycles with Phusion High-Fidelity DNA polymerase (New England Biolabs). Libraries were multiplexed for 75-bp paired-end sequencing on an Illumina NextSeq500. PBAT (BS-seq)