Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
TP53

Cell type

Cell type Class
Breast
Cell type
MCF-7
Primary Tissue
Breast
Site of Extraction
Pleura
Tissue Diagnosis
Adenocarcinoma

Attributes by original data submitter

Sample

source_name
MCF-7, NCS-treated, biological replicate 1
cell type
Breast carcinoma
cell line
MCF-7
chip antibody
p53 (Santa Cruz Biotechnology, sc-126 X)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Samples were prepared as described by Barski et al. (Cell, 2007) with minor changes. Briefly, 40,000,000 cells were cross-linked with 1% formaldehyde for 10 min. Cross-linking was stopped by the addition of glycine to 125 mM final concentration, and cells were washed twice with PBS. Cells were then harvested by scraping, and the pellet was washed once with PBS + 0.5% BSA and resuspended in RIPA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and 200 mM NaCl) supplemented with cOmplete™ protease inhibitor tablet (Roche, 11697498001) to a final concentration of 5,000,000 cells/mL. Samples were sonicated for 15 min with 20-sec pulses, 40-sec resting, using the Q700 Sonicator (QSONICA) to produce chromatin fragments of 300 bp on average. After clarification by centrifugation, sonicated extracts (other than input DNA) were subjected to immunoprecipitation. 4 µg of antibody was mixed with 40 µL of Dynabeads Protein A (ThermoFisher, 10002D) and incubated at 4°C for 6 h with rotation. Chromatin from 5,000,000 cells was added to the Protein A-antibody complexes and incubated overnight at 4°C with rotation. Immunoprecipitates were washed twice with RIPA buffer, twice with RIPA buffer + 300 mM NaCl, twice with LiCl buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate), and twice with TE buffer. The beads were then resuspended in TE buffer + 0.25% SDS + 500 µg/mL proteinase K (New England Biolabs, P8102S) and incubated overnight at 65°C. After elution, the DNA was recovered from the eluate by phenol chloroform extraction followed by ethanol precipitation in the presence of 20 µg of glycogen (Roche, 10901393001) and dissolved in TE buffer. Samples were quantified using a Quant-iT™ PicoGreen™ dsDNA Assay Kit (ThermoFisher, P11496), and fragment sizes were assessed using a Bioanalyzer (Agilent) with a High Sensitivity DNA Kit (Agilent, 5067-4626). Libraries were then prepared using TruSeq ChIP Library Preparation Kits (Illumina, IP-202-1012 and IP-202-1024) according to the manufacturer's instructions. Briefly, DNA was end-repaired, and 3’ ends were adenylated to prepare for adapter ligation. After ligation of adapters, ligation products were purified and size-selected for fragments of length 250-300 bp. Fragments with adapters were enriched by 18 cycles of PCR, then pooled for sequencing.

Sequencing Platform

instrument_model
NextSeq 500

hg19

Number of total reads
31428004
Reads aligned (%)
86.3
Duplicates removed (%)
11.8
Number of peaks
2156 (qval < 1E-05)

hg38

Number of total reads
31428004
Reads aligned (%)
88.5
Duplicates removed (%)
10.2
Number of peaks
2248 (qval < 1E-05)

Base call quality data from DBCLS SRA