GSM5861511: 356179; Homo sapiens; RNA-Seq
For RNA isolation, media was gently removed and 300μl RNA lysis Solution RA1 + 6μl TCEP (Machery-Nagel; Cat No: 740902.50) was added to each well. The lysate was collected in a 1.5ml Eppendorf tube. Each tube was vortexed vigorously and placed on ice. Further steps were carried out according to manufacturer's protocol of the Machel-Nagel RNA XS kit. RNA was checked for quality using the Agilent 4200 Tapestation. Samples with RIN > =8.5 were considered for library preparation and sequencing using the Illumina NovoSeq Total RNA is normalized to 7.5ng/ul in a total volume of 50ul on the Perkin Elmer Janus Workstation (Perkin Elmer, Janus II).  Poly-A selection and cDNA synthesis are performed using the TruSeq Stranded mRNA kit as outlined by the manufacturer (Illumina, cat#RS-122-2103).  All steps are automated on the Perkin Elmer Sciclone NGSx Workstation to reduce batch to batch variability and to increase sample throughput.  Final RNASeq libraries are quantified using the Quant-it dsDNA High Sensitivity assay, and library insert size distribution is checked using a fragment analyzer (Advanced Analytical; kit ID DNF474).  Samples where adapter dimers constitute more than 4% of the electopherogram area are failed prior to sequencing.  Technical controls (K562,Thermo Fisher Scientific, cat# AM7832), are compared to expected results to ensure that batch to batch variability is minimized.  Successful libraries are normalized to 10nM for submission to sequencing. Libraries were multiplexed and sequenced on Illumina Novaseq6000 to generate 100bp PE reads.