GSM4455045: H3K36me2_P12_GO; Mus musculus; ChIP-Seq
Ovaries were incubated in 2 mg/mL collagenaseA/0.2% trypsin/PBS at 37℃ for 10-20 minutes with serial pipetting and the same volume of M2 medium was added. Oocytes were then passed into 0.1% BSA/PBS and collected into drops of M2 medium. Oocytes with surrounding somatic cells were passed through several times into pulled glass capillaries to exclude somatic cells from oocytes and oocytes were collected in drops of M2 medium. Oocytes were then passed through 3-5 times into 0.1%BSA/PBS drops and collected. ULI-N-ChIP-seq protocol (Brind'Amour et al., 2015) was used for generating ChIP-seq libraries with some modifications. Briefly, chromatin was fragmentated by MNase (NEB), diluted in native ChIP buffer (20 mM Tris-HCl pH 8.0; 2 mM EDTA; 150 mM NaCl; 0.1% Triton X-100) containing 1 mM PMSF and EDTA-free protease inhibitor cocktail (Roche) and sonicated 10 second sonication cycles for 3 times with low power (Bioruptor). Following pre-clearing of chromatin fragments with protein A and G beads (ThermoFisher Scientific) or Dynabeads M-280 Sheep anti-Mouse IgG beads (ThermoFisher Scientific), chromatin fragments were split and incubated with each of the histone methylation antibodies at 4 degree for overnight. Following purification of ChIPed DNA by AMPure XP beads, end repair and A-tailing were performed using NEBNext UltraII End Repair/dA-Tailing Module (NEB) and ligation was carried out using NEBNext Ultra II Ligation Module (NEB) with an in-house adaptor which is compatible with Illumina sequencers. After purification of adaptor-ligated products by AMPure XP beads, libraries were amplified with unique indexing primers for 12-14 cycles. Following purification of the amplified libraries by AMPure XP beads, libraries were pooled, run on 2% E-Gel EX Gel (ThermoFisher Scientific) and the fragments from 200-600 bp was excised and purified by Zymoclean Gel DNA Recovery Kit (Zymo Research).
GEO Accession
GSM4455045