GSM4455012: H3K36me2_E16.5_PSG_Nsd1_KO1; Mus musculus; ChIP-Seq
Testes were isolated and incubated at 37℃ for 5-8 minutes in 0.25% trypsin/0.5 mM EDTA/DNaseⅠ/PBS with serial pipetting to obtain single cell suspensions. After quenching the reaction with fetal bovine serum (FBS), cells were washed once with FACS buffer (2% FBS/1 mM EDTA/PBS), stained with Propidium Iodide (PI) and passed through a 35 μm cell strainer (Falcon). FACS was carried out on a BD Influx. Following removal of doublet/triplet cells, OCT3/4-EGFP+ (PI-) cells were sorted. 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
GSM4455012