GSM4998169: PROseq_293T-WT_Rep2_R1; Homo sapiens; OTHER
For PRO-seq, 25 million wild type or mutant HEK293T cells were mixed with 2.5 million Drosophila S2 cells (10:1 ratio), permeabilized, and incubated with biotin-labeled NTPs. Biotinylated RNA was hydrolyzed with NaOH, and biotin containing RNA fragments were enriched by affinity purification using streptavidin-coated magnetic beads. A 3′ sequencing adaptor was ligated to the biotinylated RNA, the 5′ cap was removed, 5′ OH was generated by base hydrolysis and converted to 5′ phosphate by treatment with T4 polynucleotide kinase, a 5′ sequencing adaptor was ligated to RNA. RNA was purified to enrich for biotinylated fragments with adaptor at both ends and reverse transcribed. Detailed methods are in PRO-Seq, Mahat et al Nat Protoc, 11:1455-76; doi: 10.1038/nprot.2016.086.Detailed methods are in PRO-Seq, Mahat et al Nat Protoc, 11:1455-76; doi: 10.1038/nprot.2016.086. PRO-seq libraries were generated essentially as described in Mahat et al., 2016 (Nat Protoc, 11:1455-76; doi: 10.1038/nprot.2016.086). Primers RPI1, RPI2, RPI3 and RPI4 were used to barcode cDNAs from 293T-WT_Rep1 (Wild type, replicate 1), 293T-WT_Rep2 (Wild type, replicate 2), 293T-EAF1mut_Rep1 (Mutant EAF1, replicate 1), and 293T-EAF1mut_Rep2 (Mutant EAF1, replicate 2), respectively. PRO-seq libralies were pooled and sequenced as paired-end reads on the Illumina Novaseq 6000 platform.