GSM2533848: RNAseq_CN_2; Mus musculus; RNA-Seq
Cells were processed for FACS as described for HiC, except without fixation, permeabilization or fxCycle addition. GFP+ purified cells were immediately pelleted and resuspended in TRIzol (ThermoFisher, Cat.N: 15596026). After 5min at room temperature samples were vortexed for 20s, 0.2x volumes of chloroform was added, tubes were mixed by inverting and samples were centrifuged at 13,000 rpm at 4C for 15min. The aqueous phase was then processed using the RNeasy Mini Kit (Qiagen, Cat.N: 74104) according to the manufacturer's instructions. Purified RNA (maximum of 1ug) was then used for library generation using the TruSeq Stranded Total RNA Library Kit (Illumina, RS-122-2301). In brief, rRNA was depleted using RiboZero Gold, RNA was fragmented and first strand cDNA was synthesized in the presence of actinoMycin D. Second strand cDNA was then synthesised in the presence of dUTP and dsDNA was selected using AMPure XP beads. DNA was then 3'-adenylated, ligated to Illumina adapters and PCR-amplified for 10-12 cycles.