Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
FOXO1

Cell type

Cell type Class
Blood
Cell type
Pre-leukemia stem cells
NA
NA

Attributes by original data submitter

Sample

source_name
FOXO1_ ChIPseq_in_pre-leukemia_cells
cell population
CD34+ human cord blood cells
cell type
pre-leukemia stem cells

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP-Seq assays in AE cells ,were performed essentially as previously described (Ptasinska et al., 2014). Antibodies for HA (Sigma, H6908) and FOXO1 (Abcam ab39670) were used.Cells were first washed with PBS and then cross-linked with 0.83 mg/ml Di(N-succinimidyl) glutarate (DSG) (Sigma) for 45 minutes at room temperature with rotation. Cells were washed four times with PBS and further crosslinked in PBS with 1% formaldehyde (~0.34 M) for 10 minutes at room temperature. All crosslinking reactions were quenched by adding 4 volumes of PBS and 0.125 M glycine, cells were washed twice in PBS and incubated with Buffer A (10 mM HEPES pH 8.0, 10 mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100, proteinase inhibitor cocktail (Roche UK, Burgess Hill, UK) and 0.1 mM PMSF), incubated for 10 min at 4°C with rotation, and centrifuged 5 min at 500 x g at 4 °C. The pellet was resuspended in 10 ml of ice–cold Buffer B (10 mM HEPES pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.01% Triton X-100, protease inhibitor cocktail and 0.1 mM PMSF), incubated for 10 min at 4 °C with rotation and centrifuged for 5 min at 500 x g at 4 °C. Cells were resuspended in 600 μl of ice-cold ChIP lysis buffer (25 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.25% SDS, protease inhibitor cocktail and 0.1 mM PMSF), incubated 10 min on ice and sonicated at 5 °C using a Bioruptor™ (Diagenode) to generate fragments an average length of 500 bp (10 min with 30 s “ON” and “OFF” cycles, power setting high). The lysates were centrifuged for 5 min at 16,000 x g at 4 °C and the supernatants were diluted with two volumes of ice-cold ChIP dilution buffer (25 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 7.5% glycerol, protease inhibitor cocktail and 0.1 mM PMSF). For each IP, 15 μl of Dynabeads® protein G (Dynal) were pre–incubated with 50 μg BSA and 1-2 μg antibody for 2 h at 4 °C with rotation. The blocked antibody-bound protein G mix was added to 20–25 μg chromatin in a total volume of 500 μl diluted ChIP lysis buffer and incubated for 2 h at 4°C with rotation. After magnetic separation the beads were washed once with 1 ml wash buffer 1 (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), twice with 1 ml wash buffer 2 (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), once with 1 ml LiCl buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% NP-40, 0.5% Na-deoxycholate) and twice with 1 ml TE/NaCl buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM EDTA). For each wash the beads were mixed with ice-cold washing buffers for 10 min at 4 °C. The immunoprecipitated DNA was eluted two times with 50 μl ChIP elution buffer (100 mM NaHCO3, 1% SDS) for 15 min at RT with shaking. At this step the input control (1% of the starting material) was included in the experimental procedure after first adjusting the final volume to 100 μl with ChIP elution buffer. The eluted DNA was incubated overnight at 65 °C in the presence of 50 μg proteinase K. The DNA was finally purified using Agencourt AMPure (Beckman Coulter) magnetic beads according to the manufacturer’s instructions, eluted with 50 μl x TE and analysed by qPCR using SYBR Green reagents. KAPA Hyper Prep Kit, Kapa biosystems

Sequencing Platform

instrument_model
Illumina HiSeq 2500

hg19

Number of total reads
126574213
Reads aligned (%)
43.2
Duplicates removed (%)
50.9
Number of peaks
27878 (qval < 1E-05)

hg38

Number of total reads
126574213
Reads aligned (%)
44.4
Duplicates removed (%)
49.9
Number of peaks
28223 (qval < 1E-05)

Base call quality data from DBCLS SRA