GSM3744545: CBFb CD4+ T ip; Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
Cbfb
Cell type
Cell type Class
Blood
Cell type
CD4+ T cells
NA
NA
Attributes by original data submitter
Sample
source_name
Mouse CD4+ T cells
strain/background
C57Bl/6
cell type
CD4+ T cells
chip antibody
Anti-CBFb
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For ChIP-seq using CBFb Ab, 0.5x10^7 T cells were washed with ice-cold PBS three times 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 manufacturer's protocol. Libraries were sequenced to 50 bp read length on a HiSeq2000 (Illumina).