Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
EGR1

Cell type

Cell type Class
Others
Cell type
Fibroblasts
NA
NA

Attributes by original data submitter

Sample

source_name
hBJV600E_doxycycline and clobetasol_24 hours
cell line
BJ-hTert-B-RAF-V600E
cell type
human fibroblasts
inducer
doxycycline 1000 ng mL-1
treatment
clobetasol 2nM
chip antibody
anti-EGR1 Ab (sc-189X)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were the resuspended in 1mL of cold IP buffer (10 mM Tris-HCl (pH8), 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine sodium salt) and sonicated in a Covaris S220 (Brighton, UK) with the following parameters: 150W, 10%, 200 bursts/cycle, 5x2 mins. 250µL of sonicated chromatin was diluted with 500µL of cold IP buffer containing addtionnally Roche protease inhibitor cocktail. 6 µg of anti-EGR1 Ab (sc-189X) was then added for each IP and incubated O/N with rotation at 4°C. 30 µL of protein G-sepharose beads that had been blocked by incubation with IP buffer containing 2% BSA were then added for each IP and incubated for 2 hours with rotation at 4°C. The beads were then washed once consecutively with WB1 (20 mM Tris-HCl (pH8), 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 0.25% Triton x-100), WB1 with 500 mM NaCl, WB 2 (10 mM Tris-HCl (pH8), 250 mM LiCl, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Igepal CA-630) and TE (10 mM Tris-HCl (pH8), 1 mM EDTA). The washed beads were then resuspended in 200 µL of TE+ 1% SDS. 1µL (20 µg) of proteinase K solution was added and the suspension was incubated with shaking O/N at 65°C in a Thermomixer (Eppendorf) to digest proteins and to reverse the formaldehyde crosslinks. Immunoprecipitated DNA was then purified by phenol/chloroform extraction and ethanol precipitation. A DNA library for sequencing was prepared with a Diagenode IP-Star platform using their MicroPlex Library Preparation kit v2. Amplified DNA fragments with ligated sequence adapters were sized to approximately 325bp with AMPure XP beads (A63880, Beckman Coulter). The size of the final library fragments was verified on an Agilent Bioanalyzer and DNA concentrations were determined with an Invitrogen Qubit fluorimeter. Libraries were pooled in equimolar proportions and sequenced on an Illumina NextSeq500 instrument, using NextSeq 500/550 High Output 75 cycles kits; 43 (Paired-End) sequencing cycles were performed.

Sequencing Platform

instrument_model
NextSeq 500

hg19

Number of total reads
28461794
Reads aligned (%)
179.7
Duplicates removed (%)
8.2
Number of peaks
1718 (qval < 1E-05)

hg38

Number of total reads
28461794
Reads aligned (%)
181.9
Duplicates removed (%)
7.9
Number of peaks
1868 (qval < 1E-05)

Base call quality data from DBCLS SRA