GSM4987866: D19-238036; Homo sapiens; RNA-Seq
Cells were harvested by trypsinization, washed with PBS, and total RNA was extracted using the Omega RNA Purification Kit. RNA samples were quantified and quality assessed using an Advanced Analytical Fragment Analyzer. The initial steps were performed on a Tecan EVO150.10ng of totalRNA was used for library. 3'DGE-custom primers were added to a final concentration of 1 uM.   Maxima H Minus RT was then added per manufacturer's recommendations with Template-Switching oligo5V6NEXT followed by incubation at 42C for 90' and inactivation at 80C for 10'. cDNA from 12 wells containing unique well identifiers were pooled together and cleaned using RNA Ampure beads at 1.0X. cDNA was eluted with 17 ul of water followed by digestion with Exonuclease I at 37C for 30 minutes, inactivation at 80C for 20 minutes.  Second strand synthesis and PCR amplification was done by adding the Advantage 2 Polymerase Mix (Clontech) and the SINGV6 primer (10 pmol) directly to the exonuclease reaction. 15 cycles of PCR were performed followed by clean up using regular SPRI beads at 0.6X, and eluted with 20ul of EB. Successful amplification of cDNA was confirmed using the Fragment Analyzer. Illumina libraries are then produced using standard Nextera tagmentation substituting P5NEXTPT5-bmc primer in place of the normal N500 primer. Final libraries were cleaned using SPRI beads at 0.7X and quantified using the Fragment Analyzer and qPCR before being loaded for paired-end sequencing using the Illumina NextSeq500 in paired-end mode (20/50 nt reads).