GSM6198379: NTG_2; Homo sapiens; RNA-Seq
RNA from was extracted using the PureLink RNA Mini Kit (Invitrogen). Library construction was performed by the Roy J. Carver Biotechnology Center (University of Illinois, Urbana, IL, USA). Briefly, DNase-treated RNA was converted into barcoded polyadenylated mRNA libraries using the Kapa Hyper Stranded mRNA Sample Prep Kit (Roche). Libraries were barcoded with unique dual indexes to prevent index switching and adaptor-ligated double-stranded cDNAs were PCR-amplified for eight cycles with KAPA HiFi DNA Polymerase (Roche). Final libraries were quantitated by Qubit (Thermo Fisher Scientific), and the average cDNA fragment sizes were determined on a fragment analyzer. Libraries were then diluted to 10 nM and quantitated by qPCR on a CFX Connect Real-Time qPCR system (Bio-Rad) to confirm accurate pooling of barcoded libraries and to maximize the number of clusters in the flowcell. Barcoded RNA-seq libraries were sequenced using a NovaSeq 6000 on one SP lane with single-reads 100 nt in length (Illumina).