GSM3388276: IPCT-LRRM2016-52310-207697-B-TU-F01; Homo sapiens; RNA-Seq
After isolation, RNA was quantified by Picogreen (Invitrogen) and quality was assessed using the 2200 Tapestation (Agilent). RNA from each sample (10-100 ng) was converted in double stranded cDNA using Ovation RNA-Seq System V2 kit from Nugen. cDNA was sheared by sonication with the following conditions: Peak Incident Power 175, Duty Cycle 20%, Intensity 5, Cycles per Burst 200, and 120 seconds using Covaris E220 instrument (Covaris). The sheared DNA proceeded to library prep using KAPA library prep hyper kit (KAPA) following the “with beads” manufacturer protocol. At the end of the library prep, samples were analyzed on TapeStation to verify correct fragment size and to ensure the absence of extra bands. Samples were quantified using KAPA qPCR quantification kit. DNA was pooled for capture (2-6 samples per pool). We used whole exome biotin labeled probes from Roche Nimblegen (V3) and followed manufacture's protocol for the capture step. The quality of each captured sample was analyzed on TapeStation using the DNA High Sensitivity kit and the enrichment was accessed by qPCR using specific primers designed by Roche Nimblegen. The cutoff for the enrichment was 50 fold minimum.
GEO Accession
GSM3388276