GSM1186812: B-LCL-2.2; Homo sapiens; RNA-Seq
Total RNA was isolated from 5 million B-LCLs using RNeasy mini kit including DNase I treatment (Qiagen) according to the manufacturer's instructions. Total RNA was quantified using the NanoDrop 2000 (Thermo Scientific) and RNA quality was assessed with the 2100 Bioanalyzer (Agilent Technologies). Transcriptome libraries were generated from 1 μg of total RNA using the TruSeq RNA Sample Prep Kit (v2) (Illumina) following the manufacturer's protocol. Briefly, poly-A mRNA was purified using poly-T oligo-attached magnetic beads using two rounds of purification. During the second elution of the poly-A RNA, the RNA was fragmented and primed for cDNA synthesis. Reverse transcription of the first strand was performed using random primers and SuperScript II (InvitroGene). A second round of reverse transcription was done to generate a double-stranded cDNA, which was then purified using Agencourt AMpure XP PCR purification system (Beckman Coulter). End repair of fragmented cDNA, adenylation of the 3' ends and ligation of adaptors were completed following the manufacturer's protocol. Enrichment of DNA fragments containing adapter molecules on both ends was done using 15 cycles of PCR amplification and the Illumina PCR mix and primers cocktail.
GEO Accession
GSM1186812