GSM4998187: Biotinylation-seq_293T-EAF1mut_LSM11_pulldown_Rep1_R1; Homo sapiens; OTHER
For antibody-based in situ biotinylation-seq, HEK293T cells cultured in 10 cm dish were fixed with 4% PFA and permeabilized with 0.5% Triton X-100. After blocking, cells were incubated with primary antibodies, and then incubated with HRP-labeled secondary antibodies. After washing, HRP-based in situ biotinylation was performed by incubating cells with biotinylation buffer. The cells were lysed in 1% SDS RIPA buffer and were sonicated for DNA fragmentation. The biotinylated chromatin was pulled-down using SoftLinkTM Soft Release Avidin Resin (Promega), and the chromatin DNA was purified. 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.