GSM5823372: RNAseq_HeLa_PURA_siRNA_s3; Homo sapiens; RNA-Seq
Cells were washed using PBS and subsequently collected in TRIzol Reagent (Thermo Fisher Scientific). Total RNA was extracted using Maxwell® RSC miRNA Tissue Kit (AS1460) on the Maxwell® RSC instrument according to the manufacturer's instructions. Briefly, shock frozen cells were resuspended in 500 µl Trizol. 6 µl 1-Thioglycerol were added to 300 µl Trizol suspension. The suspension was mixed vigorously and total RNA extracted by phase separation using RSC SimplyRNA Tissue (AS1340) setup. Libraries were generated with the Lexogen SENSE mRNA-seq Library Prep Kit V2 (Lexogen) as described in the manual. Briefly, 100 ng of total RNA were bound to oligo-dT beads, reverse-transcribed on the beads and the second strand of cDNA was synthesized after ligation of an adapter. The eluted and adapted cDNA was amplified with barcoded primers during 11 cycles of PCR with the following program: 98 °C for 10 s, 65 °C for 20 s, 72 °C for 30 s and a final extension at 72 °C for 1 min (hold at 10 °C). The PCR products were purified with Agencourt AMPure XP beads (Beckman Coulter). The quality of the libraries was validated with an Agilent Bioanalyzer (DNA 1,000 Kit). Barcoded libraries were pooled and sequenced on an Illumina HiSeq1500 as 50 b single reads with an average sequencing depth of 20 x106 reads per sample. Each library yielded on average 35 million reads.