GSM2913910: Hela-S3-GFP Input; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Uterus
Cell type
HeLa
Primary Tissue
Cervix
Tissue Diagnosis
Adenocarcinoma
Attributes by original data submitter
Sample
source_name
HeLa-S3 cell line
cell line
HeLa-S3
shRNA status
None
chip antibody
None
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were fixed with 0.37% formaldehyde for 10 minutes at room temperature and quenched with 20 mM Glycine for 5 min. Cells were washed 2 times with cold PBS supplemented with EDTA-free Complete Protease Inhibitor cocktail from Roche (PIC), and scraped 2 times with 2.5 ml of cold PBS+PIC w/o EDTA. Pellets were suspended in 1.6 ml of RIPA buffer (50 mM Tris-HCl (pH 8), 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and PIC), sonicated in 15 ml conical tubes 3 times for 10 min at high 10 sec on/off cycles in a cooled Bioruptor and cleared by centrifugation for 15 min at 20,000 g at 4°C. The chromatin was quantified and the quality of DNA shearing was checked on a 1% agarose gel. The chromatin was diluted with RIPA Buffer to contain the equivalent of 10 ng/µl of DNA. 1 ml of chromatin corresponding to 10 µg of DNA was incubated with the appropriate antibodies O/N at 4°C, then with 25 µl of pre-washed Protein G Dynabeads® for 3 h at room temperature. The beads were washed for 5 min at RT with 1 ml of TSE-150 (0.1% SDS, 1% Triton X-100, 150 mM NaCl, 2 mM EDTA, 20 mM Tris-HCl, pH 8), TSE-500 (0.1% SDS, 1% Triton X-100, 500 mM NaCl, 2 mM EDTA, 20 mM Tris-HCl, pH 8), LiCl buffer (0.25 M LiCl, 1% NP-40, 1% DOC, 1 mM EDTA, 10 mM Tris, pH 8) and TE (10 mM Tris-HCl, pH8, 0.1 mM EDTA). The immuno-complexes were eluted from the beads using a total of 125 µl of elution buffer (100 mM sodium bicarbonate, 1% SDS) for 30 min at 30°C and de-crosslinked overnight at 65°C. The ChIP samples were purified with the Qiaquick® PCR purification kit (Qiagen) and the DNA was eluted from the columns with 50 µl of water. 10 ng of DNA (size ~200 bp) from inputs and ChIP was used per ChIP-Seq library (Bioo ChIP-Seq kit) following manufacturers instructions. 8 barcoded libraries were sequenced in one lane with a HiSeq2000 instrument to obtain single-end, 36 bp reads.