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
HeLa cells
treatment
Paraformaldehyde fixation (1%, 30 minutes)
chip antibody
none

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
25μL of ChIP-IT Protein G Magnetic Beads (ActiveMotif) were incubated with the antibody under evaluation (the amounts of antibody in use corresponded to that indicated by the supplier’s information) in presence of 100μL PIC-containing Lysis Buffer. After two hours at 4°C on a rotating shaker, chromatin from 3 million cells was added and the final vol- ume was adjusted to 500μL with PIC-containing Lysis Buffer and incubation on a rotating shaker was continued overnight at 4°C. The immunoprecipitated chromatin was recovered by magnetic bead separation, followed by multiple washing steps on a custom liquid handling platform (TECAN EVO75). Specifically, the washing is performed as follows: (1) Low salt washing (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM TrisHCl pH 8, 150mM NaCl); (2) High salt washing (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM TrisHCl pH 8, 500mM NaCl); (3) LiCl-washing (0.25M LiCl, 1% IGEPAL CA630, 1% Na-deoxycholate, 1mM EDTA, 10mM Tris pH 8) and (4) 1×TE washing. The immunoprecipitated chromatin was eluted and de-crosslinked in 100μL of elution buffer (1% SDS, 100mM NaHCO3, 250mM NaCl, 0.2mg/ml Proteinase K) and incubated for 4 hours at 65°C. The eluted chromatin was supplemented with 200μL H2O and 300μL phenol/chloroform/isoamyl alcohol (25/24/1) mix was added. After two extraction steps, the aqueous phase was subjected to ethanol precipitation in presence of 1μL GlycoBlue (Invitrogen; 15mg/ml). The precipitated material was re-suspended in 45μL H2O; 5μL was used for validation by quantitative PCR, the remaining 40μL was used for DNA library preparation. The DNA library preparation for massive parallel sequencing was performed according to standard procedures (NEXTFlex ChIP-Seq Kit (Biooscientific)) adapted to automation by our custom liquid handling platform (TECAN EVO75). Prior to DNA sequencing library preparation was monitored using a Tapestation (Agilent). Samples were sequenced on an Illumina HiSeq 2000 platform following manufacturer’s standard procedures and certified on the basis of the NGS-QC Generator concept (Mendoza-Parra et al; NAR 2013).

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg38

Number of total reads
44837267
Reads aligned (%)
99.1
Duplicates removed (%)
35.9
Number of peaks
1182 (qval < 1E-05)

hg19

Number of total reads
44837267
Reads aligned (%)
98.5
Duplicates removed (%)
37.6
Number of peaks
1272 (qval < 1E-05)

Base call quality data from DBCLS SRA