title: GSM6133521: R11345; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted from bulk cells in two steps, RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (TRIzol, Thermo Fisher Scientific, Waltham, MA, USA) and the aqueous phase was processed using Qiagen RNeasy Micro clean-up procedure (Qiagen, Hilden, Germany). Purified RNA samples were quality checked using Agilent RNA 6000 Pico assay (Agilent Technologies, Paolo Alto, CA). Reverse transcription and amplification were performed according to established method (Picelli et al., 2014). cDNA was synthesized from 2ng of purified total RNA using modified oligo(dT) primers along with 1ul of a 1: 50,000 dilution of ERCC RNA Spike in control (Ambion® Thermo Fisher Scientific). To generate sufficient quantities of cDNA for downstream library preparation steps, 13 cycles of PCR amplification was performed. The quantity and integrity of cDNA was assessed using DNA High Sensitivity Reagent Kit, Perkin Elmer LabChip GX (PerkinElmer, Waltham, MA, USA). Subsequently, pooled cDNA libraries were prepared (250 pg of cDNA per sample) using Nextera XT Kit (Illumina, San Diego, CA, USA) with dual indices for de-multiplexing. The libraries were qPCR quantified (Kapa Biosystems, Wilmington, MA) to ascertain the loading concentration. Samples were subjected to an indexed PE sequencing run of 2x151 cycles on an Illumina HiSeq 4000.