GSM1678858: Not infected. Not treated - Rep 2; Homo sapiens; RNA-Seq
Cell pellets containing 2.5 106 cells were resuspended in 1 ml of TRIZOL reagent (Invitrogen) and the RNA was purified as recommended by the manufacturer. The RNA was digested with RNAse-free DNase (1 u/mg) for 1 h at 37ºC, extracted with phenol-chloroform-isoamylalcohol and precipitated with ethanol.  The purified RNA was resuspended in nuclease free water and the absorbance was measured at 260 nm (NanoDrop ND-1000). Ribosomal RNA was removed using the Ribo-Zero Removal Kits purchased to Illumina (RZH1046). Each RNA preparation was monitored using the Agilent 2100 Bioanalyzer (Agilent technologies). Ribosomal-depleted RNA was fragmented to approximately 300bp. cDNA was synthesized using reverse transcriptase (SuperScript II, ref. 18064-014, Invitrogen) and random primers. The second strand of the cDNA incorporated dUTP instead of dTTP. Double-stranded DNA was subjected to A-tailing and ligation of the bar-coded Truseq adapters. Purification steps were done using Ampure XP Beads. Library amplification was performed by PCR using the primer cocktail supplied in the kit. Final libraries were analysed using Agilent DNA 1000 chip to estimate the quantity and check size distribution, and were then quantified by qPCR using the KAPA Library Quantification Kit (ref. KK4835, KapaBiosystems) prior to amplification with Illumina’s cBot.
GEO Accession
GSM1678858