GSM5664133: COLO 829 RNA-seq rep 2; Homo sapiens; RNA-Seq
Cells were harvested and pellets were flash frozen on dry ice. RNA was extracted using the RNeasy Mini Kit (QIAGEN). RNA-seq was performed on biological replicates using the Lexogen Quant-seq 3' mRNA-seq Library Prep Kit FWD for Illumina protocol (Lexogen, 015.96). Briefly, total RNA was extracted from cells using the RNeasy Mini Kit (QIAGEN). 1 ug of total RNA was hybridized with an oligo-dT primer containing an Illumina-compatible sequence at its 5' end and reverse transcription is performed. Following first strand cDNA synthesis, RNA is removed. Double-stranded cDNA was synthesized followed by a purification step. qPCR was performed to determine optimal PCR cycle number using the PCR Add-on Kit for Illumina (Lexogen, 020.96). i7 adapters for Illumina sequencing were added during PCR amplification. Following purification, RNA-seq libraries were quantified using the BioAnalyzer High Sensitivity DNA Kit (Agilent) prior to sequencing using 1x150 bp single-end reads on an Illumina HiSeq 4000 instrument at a depth of 50 million reads per sample.