GSM3586376: PC3: IgG 1 DMSO; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
TFs and others
Cell type Class
Attributes by original data submitter
Sequenced DNA Library
Chromatin immunoprecipitation (ChIP) was performed from ~20x106 cells by first cross-linking cells with 1% para-formaldehyde (pFa) in room temperature for 10 minutes. PFa was quenched by addition of 125mM glycine for 10 minutes and cells were washed twice in ice-cold PBS. Next, cells were solubilized into ChIP lysis buffer (0.1% SDS, 1% Triton X-100, 0.15M NaCl, 1mM EDTA, 20mM Tris pH8.0), and samples were sonicated using Bioruptor (Diagenode) to obtain DNA fragments of 200-500 based pairs in length (this was confirmed for every experiment). After sonication, samples were centrifuged and supernatant was used for ChIP. Antibodies for ChIP: RL2 #ab2739 (Abcam) and MYC AF3696 (R&D systems). ChIP was performed over-night in +4˚C, and beads were washed as follows: 2x in wash buffer 1 (0.1% SDS, 0.1% Na-deoxycholate, 1% Triton X-100, 0.15M NaCl, 1mM EDTA, 20mM HEPES pH8.0), 1x in wash buffer 2 (0.1% SDS, 0.1% Na-deoxycholate, 1% Triton X-100, 0.5M NaCl, 1mM EDTA, 20mM HEPES pH8.0), 1x in wash buffer 3 (0.25M LiCl, 0.5% Na-deoxycholate, 0.5% NP-40, 1mM EDTA, 20mM HEPES pH8.0) and 2x in wash buffer 4 (10mM EDTA, 20mM HEPES). After this, DNA was extracted using phenol-chloroform extraction and used for sequencing. Libraries were prepared according Illumina's instructions accompanying the TruSeq library preparation kit.