GSM9157753: 4DNEXT33TSI4, Homo sapiens - HCT116, DMSO control, B2 T1; Homo sapiens; RNA-Seq
Total RNA was extracted using the RNeasy Plus Mini Kit (Catalog #74134, Qiagen), as per manufacturer's protocol. Purified RNA was treated with DNase I (Catalog #M0303S, New England Biolabs) for 20 minutes, followed by heat inactivation at 75°C in the presence of 5 µM EDTA. RNA integrity was assessed at the DNA Services Facility, Roy J. Carver Biotechnology Center (University of Illinois at Urbana-Champaign), using an Agilent Bioanalyzer. All samples exhibited RNA Integrity Numbers (RIN) of 10. Ribosomal RNA was depleted using the FastSelect HMR kit (Qiagen). RNA-seq libraries were prepared with the KAPA HyperPrep Stranded mRNA Library Kit (Roche). The libraries were barcoded with Unique Dual Indexes (UDI's) which have been developed to prevent index switching. The adaptor-ligated double-stranded cDNAs were amplified by PCR for 10 cycles with the Kapa HiFi polymerase (Roche). The final libraries were quantitated with Qubit (ThermoFisher) and the average cDNA fragment sizes were determined on a Fragment Analyzer. The libraries were diluted to 10nM and further quantitated by qPCR on a CFX Connect Real-Time qPCR system (Biorad) for accurate pooling of barcoded libraries and maximization of number of clusters in the flowcell. The pooled barcoded RNA-seq libraries were sequenced on a NovaSeq X Plus instrument using V1.0 sequencing chemistry for 151 paired-end cycles.