GSM4455070: RNA_E16.5_PSG_Dnmt3l_KO1; Mus musculus; RNA-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. Total RNA extracted was from 2~3 K cells by using RNeasy micro kit (Qiagen) with on-column DNaseⅠ treatment. rRNA was depleted by NEBNext rRNA Depletion Kit (NEB) and cDNA was synthesized by NEBNext RNA First Strand Synthesis Module (NEB) and double-stranded using NEBNext UltraII Directional RNA Second Strand Synthesis Module (NEB). Following purification of double-stranded DNA by AMPure XP beads, library was constructed as described previously with modifications (Brind'Amour et al., 2015). Briefly, 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
GSM4455070