title: GSM8238350: CoilinChIP_rep1; Homo sapiens; ChIP-Seq
library_name: GSM8238350
library_construction_protocol: For antibody-based in situ biotinylation-seq, HeLa 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. For ChIP-seq, crosslinking was performed with 2 mM DSG and 1% formaldehyde. Crosslinked HEK293T cells were resuspended with hypotonic buffer and then resuspended with ChIP Lysis Buffer and subjected to mild sonication using Bioruptor Sonicator (Diagenode, Denville, NJ). The lysate was diluted and incubated with anti-Coilin antibodies (HPA027885; Atlas, Bromma, Sweden)-conjugated Protein G beads overnight at 4 ºC with rotation. The beads were washed once with low-salt buffer, once with high-salt buffer, once with LiCl buffer and twice with TE buffer. DNA–protein complexes were eluted, crosslinks were reversed, and then DNA was purified. The sequence libraries for in situ biotinylation DNA-seq were prepared using TruSeq ChIP Library Prep Kit (Illumina, San Diego, CA). The sequence libraries for in situ biotinylation RNA-seq were prepared using SMART-Seq Stranded Kit (Takara). The sequence libraries for ChIP-seq were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA).