GSM9643346: Veh_Rep2; Homo sapiens; OTHER
Plates were washed in 3 times in ice-cold PBS and cells were scraped in Lysis Buffer (pooling 12 wells per treatment) prior to isolation of nuclei. Nuclear run-on reactions were performed in duplicate on aliquots of 10 million nuclei in the presence of Biotin-11-CTP, then total RNA was isolated using TRIzol LS Reagent (Invitrogen). RNA was hydrolyzed with NaOH to yield fragments ~100 bp long and nascent RNA was enriched using M-280 Streptavidin Dynabeads (Invitrogen). Sequencing adapters were ligated onto 3' ends of nascent RNA and then a second enrichment was performed using streptavidin beads. 5' ends were de-capped (RppH) and repaired (PNK) prior to ligation of sequencing adapters and the third and final streptavidin bead enrichment. Reverse transcription was then performed and purified cDNA libraries were amplified for sequencing in PCR reactions containing Illumina TruSeq Small RNA PCR Index Primers to differentially barcode the libraries and enable multiplexing. PCR-amplified libraries were size-selected for a range of ~140–350 bp (average fragment length ~250 bp) using Agencourt AMPure XP beads (Beckman Coulter) at a volume of 1X as instructed by the manufacturer. Libraries were quantified using a KAPA Library Quantification Kit for Illumina Platforms kit (KAPA Biosystems) and pooled for sequencing on an Illumina NovaSeq instrument using 150 bp reads at the University of Colorado Anschutz Medical Campus Cancer Center Genomics Shared Resource Core Facility.