GSM4455058: DNAme_E16.5_PSG_Nsd1_Control1; Mus musculus; Bisulfite-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. Two to four thousand male germ cells spiked with 0.1 ng of lambda phage DNA (Promega) were incubated in cell lysis buffer (0.1% SDS, 1 mg/mL proteinase K, 0.1 mg/mL RNaseA) at 37℃ for 60 min followed by 98℃ for 15 min, as described previously (Shirane et al., 2013) and the lysates were subjected to bisulfite conversion by EZ DNA methylation gold kit (Zymo Research). The TACS-ligation conjugated post-bisulfite adaptor-tagging (PBAT) method (Miura et al., 2012 and 2019) was used for generating PBAT libraries.
GEO Accession
GSM4455058