GSM4977598: E13.5 male H3K27me3 rep3; Mus musculus; ChIP-Seq
PGCs were FACS sorted into cold PBS and were lysed by nuclei isolation buffer (Sigma, NUC-101). For E10.5, PGCs were pooled from different embryos. 1000 PGCs were used for each ChIP reaction. Samples were permeabilized with 0.1% Triton-X-100 /0.1% deoxycholate in PBS on ice. Samples were digested with 200 U of micrococcal nuclease (New England Biolabs, M0247S) in digestion buffer at 21 °C for 7.5 mins and the reaction was stopped with 100 mM EDTA. An aliquot of chromatin (10%) was used as an input control. Samples were pre-cleaned by protein A/G beads for 2 hours at 4°C. Meanwhile, 250 ng anti-H3K9me3 (Diagenode, C15410056) and anti-H3K27me3 (Millipore, 07-449) antibodies were incubated with protein A/G beads for 2 hours at 4°C. Chromatin samples were incubated with antibody-bound beads overnight at 4°C. Samples were washed by two low-salt washes and one high salt wash and eventually DNA was eluted from the beads for 1.5 hours at 60°C. For sizes selection, AMPure XP beads were used at 1.8:1 ratio. Library preparation was performed using a MicroPlex Library Preparation kit v2 (Diagenode, C05010013) following manufacturer's recommendations. Libraries were further purified by AMPure XP beads at 1:1 ratio. Library quality was measured with a 2100 Bioanalyzer Instrument (Agilent) and library size was assessed with a Kapa library qPCR quantification kit (Roche, KK4824). Library sequencing was performed in a 100bp paired-end mode with a HiSeq 2500 System (Illumina) following manufacturer's recommendations.