GSM4346181: dC a-EBF1 A-MuLV; Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
Ebf1
Cell type
Cell type Class
Embryo
Cell type
Embryonic liver
NA
NA
Attributes by original data submitter
Sample
source_name
fetal liver EBF1 fl/fl
cell type
A-MuLV transformed pro-B cells
transfection construct
pMys_EBF1_dC_IRES_GFP
differentiation stage
pro-B
strain
C57BL/6
chip antibody
a-EBF1 (1C) rabbit antibody (Roessler et al. 2007)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
EBF1 ChIP was performed according to Boller et al. 2016 protocol with minor changes. Only crosslinking with formaldehyde was used. 4 µg rabbit a-EBF1 (1C) rabbit antibody per sample was used. Cells were harvested and washed with PBS. Cell pellet was resuspended in PBS with 2% FCS (4 mln cells per 1 ml buffer). Fresh formaldehyde mix (50mM HEPES-KOH pH8.0, 100mM NaCl, 0.5mM EGTA, 1mM EDTA, 11% (v/v) formaldehyde) was added to the cell suspension to a final concentration of 1% FA. Tubes were incubated on rotator for 10 min at RT. Quenching was performed by adding 2 M Glycine to the final concentration 0.2 M. Cell pellet was resuspended in Lysis buffer (50mM Tris-HCl pH 8.0, 1% (w/v) SDS, 10mM EDTA; PIM) (4 mln cells per 100 μl buffer). Chromatin was sheared the chromatin with a Bioruptor (20-25 cycles, output level "High", 30 sec ON, 30 sec OFF, 4°C). Fragment size and amount of chromatin was checked. 10-20 μg DNA was used for 1 ChIP reaction. Chromatin was mixed with 4 μg of EBF1 antibody or rabbit IgG and incubated on rotator ON at 4°C. Chromatin was incubated with 30 μl of Dynabeads Protein G (ThermoFisherScientific) suspension per sample on rotator for 4 hours at 4°C. Beads were washed 4 times with Wash buffer A (20mM Tris-HCl pH 8.0, 150mM NaCl, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 2mM EDTA); once with Final wash buffer A (20mM Tris-HCl pH 8.0, 500mM NaCl, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 2mM EDTA). Every wash was on rotator for 5 minutes at 4°C. Chromatin was eluted with 100 μl Elution buffer (10 mM Tris-HCl, pH 8.0; 0.5% SDS; 300 mM NaCl; 5 mM EDTA pH 8.0) by shaking vigorously at 65°C for 30 min. Samples were revese crosslinked, treated with RNase A and Proteinase K, and purified with QIAquick PCR Purification Kit. Libraries were prepared with the NEBNext Ultra II DNA Library Prep Kit for Illumina according to the manufacturer's protocol. No size selection was used. Amplification was performed using 10-12 PCR cycles. Deep sequencing was performed on a HiSeq 3000 system according to the standard Illumina protocol for 75 or 100 bp paired-end reads.