title: GSM5252986: PROseq_siNELFE_Rep1; Homo sapiens; OTHER
library_construction_protocol: 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. Ribo-depleted RNA-seq libraries were prepared using the TruSeq Stranded Total RNA Library Prep Gold (Illumina) following manufacturer's instructions. Libraries were checked for quality and quantity using Bioanalyzer. Equimolar concentrations of libraries were pooled, requantified and sequenced as 100 bp paired-end reads on the Illumina HiSeq 3000 instrument. Libraries for ChIP-seq and antibody-based in situ biotinylation-seq were prepared using TruSeq ChIP Library Prep Kit (Illumina) and IDT for illumina-TruSeq RNA UD Index (Illumina). Library purification and size selection were performed using Agencourt AMPure XP system. Libraries were checked for quality and quantity using Bioanalyzer. Equimolar concentrations of libraries were pooled, and sequenced as paired-end reads on the Illumina Novaseq 6000 platform (ChIP-seq) or Hiseq 2500 platform (antibody-based in situ biotinylation-seq). 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. 4C-seq libraries werec generated essentially as described in Krijger et al., 2020 (Methods, 170: 17-32; doi: 10.1016/j.ymeth.2019.07.014). 4C-seq libralies were pooled and sequenced on Illumina Hiseq 2500 platform.
attributes.tag: GEO Accession
attributes.value: GSM5252986