Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
Epitope tags

Cell type

Cell type Class
Blood
Cell type
2B4
NA
NA

Attributes by original data submitter

Sample

source_name
2B4 cell line
cell line
2B4
chip antibody
Anti-Flag M2
transfection
Mock gRNA

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For ChIP-seq using anti-Flag (Sigma, F3165), 1x10^7 2B4 cells were washed with ice-cold PBS three times and and were crosslinked by incubation with 1% formaldehyde containing 0.5 mM PMSF and protease inhibitor cocktail (Roche, 4693116001) on ice for 10 minutes. The crosslinking reaction was stopped by the addition of glycine to 0.125 M. Cells were incubated on ice for 5 minutes and then washed three times with ice-cold PBS. Cells were lysed by the incubation for 10 min on ice in 0.5 ml of 50 mM HEPES (pH 7.5), 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP40, 0.25% TritonX-100 containing protease inhibitor cocktail. Nuclei were pelleted and lysed by resuspending in 10 mM Tris-Cl (pH 8.0), 200 mM NaCl, 1 mM EDTA and 0.5 mM EGTA containing protease inhibitor cocktail. Pelleted chromatin was resuspended in 400 μl of 10 mM Tris-Cl (pH 8.0), 300 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% TritonX-100, 0.1% sodium deoxycholate and 0.5% N-laurylsarcosine containing protease inhibitor cocktail, and was sonicated using a model XL2000 ultrasonic cell disruptor (MICROSON) to approximately 350 bp of DNA fragments. Sonicated chromatin was incubated at 4 °C overnight with anti-Flag antibody preconjugated to magnetic beads (Dynabeads). Beads were washed by RIPA buffer 6 times and DNA was eluted and reverse-crosslinked at 65 °C in 50 mM Tris-Cl (pH 8.0), 10 mM EDTA, 1% SDS overnight. Library construction was conducted by using NEBNext ChIP-seq Library Prep Reagent Set for Illumina according to the manufacture's protocol. Libraries were sequenced to 50 bp read length on a HiSeq2000 (Illumina).

Sequencing Platform

instrument_model
Illumina HiSeq 2000

mm10

Number of total reads
41370989
Reads aligned (%)
97.5
Duplicates removed (%)
36.7
Number of peaks
536 (qval < 1E-05)

mm9

Number of total reads
41370989
Reads aligned (%)
97.3
Duplicates removed (%)
36.8
Number of peaks
585 (qval < 1E-05)

Base call quality data from DBCLS SRA