GSM5515675: PreD_M2_A3_B5_S190; Mus musculus; RNA-Seq
Skin were trypsinized to extract basal stem cells, which were then FACS-sorted with a 100-μm nozzle into 96 well PCR plates (Bio-Rad) containing 2μl of lysis buffer [0.2% Triton X-100, 2U/μl RNaseOUT (Thermo Scientific), 0.25μM oligo-dT30VN primer and 1:8,000,000 diluted ERCC spike-in RNAs (Ambion)]. Briefly, after sorting, plates were briefly centrifuged, snap-frozen in liquid nitrogen and stored at −80°C. Plates were then thawed on ice and incubated for 3 min at 72°C to lyse cells. Reverse transcription (4U/μl Maxima H- transcriptase), template switching reaction and PCR pre-amplification (15 cycles) were performed according to the protocol. PCR product was cleaned up using 0.8X AMPure XP beads (Beckman Coulter) and the quality and quantity of cDNA libraries were measured by Agilent 2200 TapeStation and Qubit Florometer (Thermal Fisher Scientific). To exclude empty wells and low-quality libraries, the cDNA libraries were assessed by qPCR with a primer pair for the housekeeping gene Gapdh (Table S2). Samples showing high Ct value (> 35 cycles) or no PCR amplification were discarded. 50-100pg of each cDNA library was used for generating Illumina sequencing library using a Nextera XT DNA library preparation kits (Illumina). After the final PCR amplification, samples were pooled and cleaned by 0.9X AMPure XP beads.