Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
GRHL2

Cell type

Cell type Class
Breast
Cell type
T-47D
Primary Tissue
Breast
Tissue Diagnosis
Adenocarcinoma Ductal

Attributes by original data submitter

Sample

source_name
T47D
cell line
T47D
cell type
Mammary gland ductal carcarcinoma epithelial cells derived from metastatic site
cell source
ATCC #HTB-133
treatment
stimulation with E2
chip antibody
Sigma Aldrich HPA004820

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
The ChIP-exo 5.0 experiment was performed using the published protocol (Rossi et al. Nat Comm 2018) with minor adaptations in regards to chromatin preparation and the IP step. Cells were grown to 80% confluence on 150cm plates and washed once with PBS and fixed with 1% formaldehyde (Thermo Fisher #28908) in 10 ml PBS for 10 min. To quench the cross-linking reaction, 1/20 volume of 2.5 M glycine was added to the plate for a final concentration of 0.125M for 5 min. The supernatant was aspirated, and the cells were washed twice with 5 ml PBS and harvested on ice using cell scrapers into 15ml sonication tubes (Diagenode cat# C01020031). Cells were pelleted at 1000g for 5 min at 4°C, washed 1x with 5 ml cold PBS, and pelleted once more. Cell pellet collected from 6 plates of cells was resuspended in 3 ml of Lysis Buffer 1 (50 mM HEPES-KOH, pH 7.5; 140 mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100), allowed to rotate for 10 min at 4°C, and pelleted at 1000 x g for 5 min at 4°C. Cells were then resuspended in 3 ml of Lysis Buffer 2 (10 mM Tris-HCL pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), allowed to rotate for 10 min at room temperature, and pelleted at 1000 x g for 5 min at 4°C. Cells were then resuspended in 3 ml of Lysis Buffer 3 (10 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine) and then sonicated on a Bioruptor Pico at 4°C for 20min of 30'' ON-OFF cycles with 1.2 g of sonication beads. After sonication, samples were transferred to 2 separate 1.5 ml microtube and centrifuged at 20, 000 x g for 15 minutes at 4°C. The supernatant was transferred to new 15 ml tubes. To check sonication efficiency, 20µl of sonicated samples was transferred to a new microtube with 80µl PBS and incubated at 65°C for 3hr on an Eppendorf Thermomixer shaking at 1000rpm to decrosslink. DNA was purified with the QIAquick PCR Purification Kit (Qiagen cat# 28106) and eluted in 50µl EB buffer. 1 ug of decrosslinked chromatin from each sample were run in a 1% agarose gel at 100V for 45min and imaged on a Bio-rad ChemiDoc XRS+. DNA concentration in the sonicated decrosslinked samples were measured via qubit, and this concentration was then used to estimate the concentration of chromatin in the un-decrosslinked samples. 120 ug of total chromatin were transferred equally to two 1.5ml LoBind tubes (Eppendorf cat# 0030108051) and each was brought up to a final volume of 500µl with Lysis Buffer 3. 10 ug of antibodies were use for transcription factor ChIP (see Key Resources table for list of antibodies), which was divided equally into two and individually pre-bound to 10 ul of Protein A Mag Sepharose Beads (GE Healthcare 28944006). The beads were washed in 1 ml of 0.5% BSA in 1x PBS three times before and after overnight pre-binding at 4°C. The chromatin samples were added to the pre-bound Dynabeads and rotated end-to-end overnight at 4°C. The beads were collected on the magnetic rack and sequentially washed with 500 uL FA Lysis Buffer, NaCl Buffer, LiCl Buffer, and 10 mM Tris-HCl buffer pH 8.0, all at 4°C. The rest of the reaction and the library preparation were performed exactly as described in the previously cited paper. Library preparation was performed as described (Rossi et al. nat Comm 2018) with minor adaptations in regards to the oligonucleotides used. NEBNext Multiplex Oligos for Illumina Set 1 was used in place of ExA2_iNN. NEBNext Universal PCR Primer for Illumina was used instead of ExA1-58. All other oligos were synthesized from Sigma except for ExA1-SSL_N5, which was ordered from IDT with 5 random basepairs at the beginning of the oligo.

Sequencing Platform

instrument_model
NextSeq 500

hg19

Number of total reads
61430480
Reads aligned (%)
51.9
Duplicates removed (%)
13.0
Number of peaks
22301 (qval < 1E-05)

hg38

Number of total reads
61430480
Reads aligned (%)
52.9
Duplicates removed (%)
12.1
Number of peaks
22416 (qval < 1E-05)

Base call quality data from DBCLS SRA