RP-2423_16466_27_102820 RNA
Total RNA was assessed for quality using the Caliper LabChip GX2.  The software calculated the percentage of fragments with a size greater than 200nt (DV200). An aliquot of 200ng of RNA was used as the input for first-strand cDNA synthesis using Illumina's TruSeq RNA Access Library Prep Kit.  Synthesis of the second strand of cDNA was followed by indexed adapter ligation. Subsequent PCR amplification enriched for adapted fragments. The amplified libraries were quantified using an automated PicoGreen assay. 200ng of each cDNA library, not including controls, were combined into 4-plex pools. Capture probes that target the exome were added and hybridized for the recommended time.  Following hybridization, streptavidin magnetic beads were used to capture the library-bound probes from the previous step.  Two wash steps effectively removed any nonspecifically bound products.  This same hybridization, capture and wash steps were repeated to assure high specificity. A second round of amplification enriched the captured libraries.  After enrichment, the libraries were quantified with qPCR using the KAPA Library Quantification Kit for Illumina Sequencing Platforms and then pooled equimolarly.  The entire process was in 96-well format, with all pipetting done by Agilent Bravo or Hamilton Starlet.     Pooled libraries were normalized to 2nM and denatured using 0.1 N NaOH before sequencing. Flowcell cluster amplification and sequencing were performed according to the manufacturer's protocols using HiSeq 2000 or 2500. Each run was a 76bp paired-end with an eight-base index barcode read.