GSM3242980: Rao-2017-CHIP039-input-treated; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Unclassified
Antigen
Unclassified
Cell type
Cell type Class
Digestive tract
Cell type
HCT 116
Primary Tissue
Colon
Tissue Diagnosis
Carcinoma
Attributes by original data submitter
Sample
source_name
Colorectal carcinoma
cell line
HCT-116-RAD21-mAC
protocol
ChIP-Seq
treatment
500uM auxin (360 min)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP-Seq for H3K27Ac, H3K4me1, H3K4me3, H3K36me3, H3K27me3, H3K9me3, H4K16Ac, H4K20me3, H3K79me2, and H2.AZ was performed using a native ChIP-Seq protocol. Chromatin from untreated HCT-116 RAD21-mAC cells or cells treated for 6 hours with 500uM IAA was digested with Mnase (Sigma) in digestion buffer (50 mM Tris-HCl, pH7.6, 1 mM CaCl2, 0.2% Triton X-100, butyrate 5 mM) for 5' at 37°C and dialyzed against RIPA buffer for 2hrs at 4°C. Five microgram of respective antibody was incubated with 40 μl of Dynabeads Protein A (or G) for 40 min at room temperature. Antibody-bound beads were added to 500 μl of sonicated or Mnase-digested chromatin, incubated at 4°C overnight, and washed twice with RIPA buffer, twice with RIPA buffer containing 0.3M NaCl, twice with LiCl buffer (0.25 M LiCl, 0.5% Igepal-630, 0.5% sodium deoxycholate), once with TE (pH 8.0) plus 0.2% Triton X-100, and once with TE (pH 8.0). ChIP DNA was purified by phenol-chloroform extraction followed by ethanol precipitation. Libraries were prepped for Illumina sequencing and 50bp single-end reads were sequenced on a HiSeq2000 or 2500 (Illumina). We also performed ChIP-Seq for RAD21 and CTCF following the same protocol as above, except that cells were fixed with 1% formaldehyde for 10 minutes at 37°C and fixation was quenched by the addition of glycine to a final concentration of 125mM for 10 minutes. The fixed cells were sonicated using a Branson sonifier at amplitude 35%, 12 cycles of 20 seconds of sonication and 30 seconds of pause. Antibody was added to the sonicated chromatin as above and the samples were further processed as above. We also performed ChIP-Seq for SMC1 and an additional replicate for CTCF following the protocol outlined by the ENCODE consortium (2012). standard Illumina library construction protocol, Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation DNA was PCR amplified with Illumina primers for 8-12 cycles and library fragments of 400-600 bp (insert plus adaptor and PCR primer sequences) were purified using SPRI beads. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the HiSeqX Ten, NextSeq 500, or HiSeq 2500 following the manufacturer's protocols