GSM5321622: P1170LM; 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.