GSM6475164: FVBL; Mus musculus; RNA-Seq
Single cell RNA-seq: single CD49fhiEpCAMmedLin- cell from different age mice or tumor was directly sorted into 96-well plate containing lysis buffer using FACS. Single cell was immediately lysed at 70 degree for 3min in the PCR system. Single cell RNA libraries were prepared for sequencing using 3' UTR Smartseq protocols. The sample was reverse transcribed to cDNA using SuperScriptIIreverse transcriptase (invitrogen, 18064-071) with a template switch oligo (TSO) primer and a sample-specific 25 nt oligo dT reverse transcription primer. Then the cDNA was amplified by 16 cycles of PCR with 3'P2 primer and IS primer using KAPA HiFi HotStart Ready Mix (Kapa Biosystem, KK2602). The barcoded DNAs were amplified using biotinylated pre-index primers. After purified by AMpure XP beads, cDNA was sonicated to approximately 300 bp fragments using Covaris ME220.  3' terminal of the cDNA was enriched using Dynabeads® MyOne Streptavidin C1 beads (Invitrogen, 65001). The RNA-seq libraries were constructed using the Kapa Hyper Prep Kit (Kapa Biosystem, KK8504) according to the manufacturer's instructions.