title: GSM5121210: Setd1b_WT_GV_H3K4me3_2; Mus musculus; ChIP-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 ChIP-seq protocol was performed. Approximately 200 GV oocytes were collected for three biological replicates of each genotype for H3K4me3 ULI-nChIP-seq. Samples were thawed on ice and permeabilized using nuclei EZ lysis buffer (Sigma) and 0.1% Triton-X-100 /0.1% deoxycholate. Micrococcal nuclease digestion was completed with 200 U of micrococcal nuclease (New England Biolabs) at 21 °C for 7.5 min. Chromatin samples were precleared in complete immunoprecipitation buffer with Protein A/G beads rotating for 2 hours at 4 °C. For each sample, 125ng of anti-H3K4me3 antibody (Diagenode, C15410003) was bound to Protein A/G beads in complete immunoprecipitation buffer for 3 hours rotating at 4 °C. Chromatin was then added to the antibody-bound beads and samples were rotated overnight at 4 °C. Chromatin-bound beads were washed with two low-salt washes and one high-salt wash and DNA was then eluted from the beads at 65 °C for 1.5 hours. Eluted DNA was purified with solid-phase reversible immobilization (SPRI) purification with Sera-Mag carboxylate-modified Magnetic SpeedBeads (Fisher Scientific) at a 1.8:1 ratio. Library preparation was completed using the MicroPlex Library Preparation kit v2 (Diagenode) with indexed adaptors, as per the manufacturers guidelines. Libraries were multiplexed for 75 bp Single End sequencing on a NextSeq500.