title: GSM3018657: Sequential_ChIPseq_FLAG-Rbbp4; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were crosslinked with formaldehyde treatment and chromatin fragmented to 200-300 bp by sonication. For sequential ChIP, 4x107 cells were lysed, and cross-linked DNA was subjected to immunoprecipitation with 50 ul of M2-Flag agarose (Sigma). Agarose beads were washed twice with RIPA buffer, re-suspended in 100 ul TE, followed by elution of the immunocomplexes with 3 ul of 5 g/ul FLAG peptide at 4°C for 2 hours. The supernatant was transferred and re-immunoprecipitated with Rbbp4 antibody (Abcam, ab79416) immobilized to Protein A beads (Dyna) before crosslinking was reversed and protein digested with Proteinase G. ChIP material was blunt-ended and phosphorylated with the End-it-Repair kit (EPICENTRE). Illumina genome sequencing adaptors were ligated with T4 DNA ligase (New England Biolabs) after addition of adenosine nucleotides, using exo-Klenow. Samples were PCR amplified with multiplexed Illumina genomic DNA sequencing primers. PCR products (250 to 450 bp in size) were gel-purified and submitted for Illumina deep sequencing.
attributes.tag: GEO Accession
attributes.value: GSM3018657