Antigen

Antigen Class

Input control

Antigen

Input control

Cell type

Cell type Class

Gonad

Cell type

Oocytes

MeSH Description

Female germ cells derived from OOGONIA and termed OOCYTES when they enter MEIOSIS. The primary oocytes begin meiosis but are arrested at the diplotene state until OVULATION at PUBERTY to give rise to haploid secondary oocytes or ova (OVUM).

Sample

source_name

p12 growing oocyte

strain background

C57BL/6

developmental stage

postnatal day 12

genotype/variation

WT

chip antibody

none

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

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).

Sequencing Platform

instrument_model

NextSeq 500

mm10

Number of total reads

18861137

Reads aligned (%)

83.8

Duplicates removed (%)

11.4

Number of peaks

114 (qval < 1E-05)

mm9

Number of total reads

18861137

Reads aligned (%)

83.8

Duplicates removed (%)

11.6

Number of peaks

97 (qval < 1E-05)