title: GSM6051728: Sm1_PROseq-APM1; Homo sapiens; OTHER
library_construction_protocol: Plates were washed in PBS and cells were scraped in Lysis Buffer prior to isolation of nuclei. Nuclear run-on reactions were performed 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 then pooled and sequenced on an Illumina NextSeq instrument using 75 bp single-end reads.