GSM8086008: RNA-seq_RKO_DMSO control_rep3; Homo sapiens; RNA-Seq
TRIzol Reagent (Invitrogen) was added directly to adhered cells, collected, and stored at -80°C. After thawing, total RNA was extracted following the manufacturer's protocol, and RNA was resuspended in DEPC Nuclease-Free water. Libraries were prepared by the Van Andel Institute Genomics Core (RRID:SCR_022913) from 500 ng of total RNA using the KAPA RNA HyperPrep Kit (Kapa Biosystems, Wilmington, MA USA). Ribosomal RNA material was reduced using the QIAseq FastSelect –rRNA HMR Kit (Qiagen, Germantown, MD, USA). RNA was sheared to 300-400 bp. Prior to PCR amplification, cDNA fragments were ligated to IDT for Illumina TruSeq UD Indexed adapters (Illumina Inc, San Diego CA, USA). Quality and quantity of the finished libraries were assessed using a combination of Agilent DNA High Sensitivity chip (Agilent Technologies, Inc.) and QuantiFluor® dsDNA System (Promega Corp., Madison, WI, USA).  Individually indexed libraries were pooled and 50 bp, paired end sequencing was performed on an Illumina NovaSeq6000 sequencer to an average depth of 50M raw paired-reads per transcriptome. Base calling was done by Illumina RTA3 and output of NCS was demultiplexed and converted to FastQ format with Illumina Bcl2fastq (v1.9.0).