GSM9022902: MCF-7_PRO-seq_DMSO 30min_rep2; Homo sapiens; OTHER
Nuclei were purified, run-on assay performed, and biontin-labeled RNA extracted by Trizol LS reagent followed by binding to streptavidin-conjugated dynabeads as described. PRO-seq libraries were generated as previously described (Judd J, et al., BioRxiv 2020). Total RNA was then enriched for biotin-labeled nascent transcripts by streptavidin-conjugated dynabead. Further fragmentation, barcoding of RNA fragments, and first strand synthesis was performed as described. The libraries were subjected to quality control analyses, such as optimizing the number of PCR cycles required to amplify each set of libraries, adapter cleanup by SPRI-bead purification, quantifying the final library yield by Qubit fluorometer (Invitrogen, Q32851), and checking the size distribution of the final library by DNA ScreenTape system (Agilent Technologies, G2964AA, 5067–5584, and 5067–5603). The libraries were barcoded with indices as described using RPI-n indexed barcodes and sequenced using Illumina NextSeq (2000) by 100 bp single-end sequencing.