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 cells
cell line
Epithelial carcinoma
chip antibody
none
cell cycle
S7

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
TAP- or LAP -tagged chromatin were purified in two steps. In the first step native TAP-tagged chromatin was immunoprecipitated by incubating the bulk soluble mononucleosome pool with rabbit IgG (Sigma-Aldrich) coupled to Dynabeads M-270 Epoxy (Thermo Fisher Scientific, 14301). Alternatively, CENP-ALAP chromatin was immunoprecipitated using mouse anti-GFP antibody (clones 19C8 and 19F7, Monoclonal Antibody Core Facility at Memorial Sloan-Kettering Cancer Center, New York) (Heiman et al., 2008) coupled to Dynabeads M-270 Epoxy. Chromatin extracts were incubated with antibody bound beads for 16 h at 4 ÅãC. Bound complexes were washed once in buffer A (20 mM HEPES at pH 7.7, 20 mM KCl, 0.4 mM EDTA and 0.4 mM DTT), once in buffer A with 300 mM KCl and finally twice in buffer A with 300 mM KCl, 1 mM DTT and 0.1% Tween 20. In the second step, TAP-chromatin complexes were incubated 16 h in final wash buffer with 50μl recombinant TEV protease, resulting in cleavage of the TAP tag and elution of the chromatin complexes from the beads. Alternatively, CENP-ALAP chromatin was eluted from the beads by cleaving the LAP tag using PreScission protease (4 h, 4ÅãC). For CENP-ATAP co-IP experiments, bound complexes were washed and proteins associating with CENP-ATAP were denatured by boiling in sample buffer, run on SDS-PAGE and blotted for CAF1 subunits and MCM2. ChIP libraries were prepared following Illumina protocols with minor modifications (Illumina, San Diego, CA). To reduce biases induced by PCR amplification of a repetitive region, libraries were prepared from 80-100 ng of input or ChIP DNA. The DNA was end-repaired and A-tailed and Illumina Truseq adaptors were ligated. Libraries were run on a 2% agarose gel. Since the chromatin was digested to mononucleosomes, following adaptors ligation the libraries size was 250-280 bp. The libraries were size selected for 200-375 bp. The libraries were then PCR-amplified using only 5-6 PCR cycles since the starting DNA amount was high. Resulting libraries were sequenced using 100 bp, paired-end sequencing on a HiSeq 2000 instrument per manufacturer's instructions with some modifications (Illumina, San Diego, CA).

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg38

Number of total reads
64049567
Reads aligned (%)
96.5
Duplicates removed (%)
3.5
Number of peaks
1961 (qval < 1E-05)

hg19

Number of total reads
64049567
Reads aligned (%)
95.5
Duplicates removed (%)
3.8
Number of peaks
592 (qval < 1E-05)

Base call quality data from DBCLS SRA