GSM2011519: Wild Type_2; Homo sapiens; RNA-Seq
The cells were dissolved in the Trizol reagent (Invitrogen; Carlsbad, CA) 24-hours post-transfection to extract total RNA. Total RNA was used for the preparation of next-generation messenger RNA (mRNA) sequencing libraries with the TruSeq RNA Sample Preparation v2 kit (Illumina) according to manufacturer's instructions. Briefly, four µg of total RNA was used for polyA mRNA selection using streptavidin-coated magnetic beads followed by thermal fragmentation of selected mRNA. The fragmented mRNA was used as a template for cDNA synthesis by reverse transcriptase with random primers. The cDNA was further converted into double stranded DNA and end-repaired to incorporate specific index adapters for multiplexing. The quality of RNA-Seq libraries was examined by qPCR and the DNA high sensitivity chip on Agilent 2100 Bioanalyzer (Agilent; Palo Alto, CA) according to manufacturer’s instructions. The libraries having unique index sequences were pooled in equimolar ratio for a final size selection (400-500bp) on a Caliper Lab XT DNA 750 chip (Caliper life Sciences, Hopkinton, MA).