title: GSM5121217: Oocytes_-_Setd1b_KO_Lib_3; Mus musculus; Bisulfite-Seq
library_construction_protocol: Ovaries were collection from 21-day-old females and digested with 2mg/mL collagenase (Sigma) and 0.02% trypsin solution, agitating at 37°C for 30 min.  GV oocytes were collected in KSOM medium (Sigma) and washed with PBS from females with an oocyte conditional deletion of Setd1b using Gdf9-cre (Setd1b cKO) and females carrying floxed Setd1b (Setd1b WT) and stored at -80C until PBAT protocol was performed. Approximately 100 GV oocytes were collected for three biological replicates of each genotype. Cells were lysed with 0.5% SDS in EB buffer and bisulphite treated with the Imprint DNA Modification kit (Sigma). The resulting DNA was purified using columns and reagents from the EZ DNA Methylation Direct kit (Zymo Research). First-strand synthesis was performed with Klenow Exo– enzyme (New England Biolabs) using a customized biotin-conjugated adaptor containing standard Illumina adaptor sequences and 9 bp of random sequences (9N), as previously described (Hanna et al., 2018). Following exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (Thermo Fisher Scientific), second-strand synthesis was performed with Klenow Exo– enzyme (New England Biolabs) using a customized adaptor containing standard Illumina adaptor sequences and 9 bp of random sequences.  Ten PCR cycles with Phusion High-Fidelity DNA polymerase (New England Biolabs) were used for library amplification with indexed adaptors.  Libraries were multiplexed for 100-bp paired-end sequencing on an Illumina HiSeq 2500.