GSM3625409: 66_A1; Homo sapiens; RNA-Seq
Following 4 days of RA treatment, NSCs were lysed directly with RLT Lysis Buffer (Qiagen) and stored at -80°C until used for RNA isolation with the RNEasy 96 kit (Qiagen) according to the manufacturer's instructions. To prepare cDNA, the Ligation Mediated RNA sequencing library protocol was performed (Hou et. al. 2015).  Briefly, mRNA was isolated from purified 100 ng total RNA using oligo-dT beads (NEB). Isolated mRNA was fragmented in reverse transcription buffer and reverse transcribed with SmartScribe reverse transcriptase (Clontech).   RNase A and RNase H treatments were then performed to remove RNA. The 5' adapter was ligated to the cDNA using RNA ligase 1 (NEB) and purified using Ampure XP Beads (Beckman).  The cDNA was then amplified with 18 cycles of PCR.  The indexed cDNA libraries were normalized and pooled at thirty samples per lane and sequenced on an Illumina HiSeq 2500 with a single 51bp read and a 10bp index read.
GEO Accession
GSM3625409