GSM8490329: patient 24; Homo sapiens; RNA-Seq
SureSelect XT HS2 RNA Target Enrichment System (Agilent Inc) 200ng of RNA was fragmented and converted to cDNA followed by end repair, duplex UMI adaptor ligation, and 11 cycles of PCR to complete the dual- index adapters. Unique dual-indexed, molecular-barcoded libraries were purified with AMPureXP beads (Beckman Coulter) and validated for appropriate size (225-275bp) on a Tapestation 4200 DNA1000 screentape (Agilent Inc.). 200ng of purified library was then hybridized to the SureSelectXT Human All Exon V8+UTR Capture library (Agilent Inc.). The hybridized regions were then bound to streptavidin magnetic beads and washed to remove any non-specific bound products. Eluted library underwent a second 13 cycle PCR amplification to generate enough material for sequencing. Final libraries were purified, measured by Tapestation 4200 DNA1000 screentape (250-350bp), and quantitated using KAPA qPCR (KAPA Biosystems). Individual libraries were pooled in equimolar 2nM final concentration. Each pool is denatured and diluted to 200pM with 1% PhiX control library added. The resulting pool is then loaded into the appropriate NovaSeq Reagent cartridge, as determined by the number of sequencing cycles desired, and sequenced on a NovaSeq6000 following the manufacturer's recommended protocol (Illumina Inc.).