Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Unclassified
Antigen
Unclassified

Cell type

Cell type Class
Blood
Cell type
Th2 Cells
MeSH Description
A subset of helper-inducer T-lymphocytes which synthesize and secrete the INTERLEUKINS IL-4; IL-5; IL-6; and IL-10. These cytokines influence B-cell development and antibody production as well as augmenting humoral responses.

Attributes by original data submitter

Sample

source_name
human naïve CD4+ T-cells differentiated toward Th1 and Th2 until day 6
cell type
Th2
biological replicate
1
file identifier
GRS-79

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
To fix the DNA-protein complex, cells were cross-linked with 1/10 volume of cross-linking solution (50 mM HEPES-KOH [pH 7.5], 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 11% formaldehyde) for 10 min followed by quenching with 1/20 volume of 2.5 mM glycine solution for 10 min. Cells were harvested and washed twice with ice-cold PBS and pelleted, and nuclear fractions were prepared. The cell pellet was dissolved in 20 ml lysis buffer 1 (50 mM HEPES KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Nonidet P-40, and 0.25% Triton X-100 with protease inhibitor Complete Mini [Roche]) and incubated for 10min at 4°C followed by centrifugation at 2000 × g for 10 min at 4°C. The cell pellet was resuspended in 10 ml lysis buffer 2 (200 mM NaCl, 1 mM EDTA [pH 8], 0.5 mM EGTA, and 10 mM Tris [pH 8], with protease inhibitor) at room temperature for 10 min. Nuclei were pelleted at 2000 × g for 10 min at 4°C and resuspended in 1ml lysis buffer 3 (1 mM EDTA [pH 8], 0.5 mM EGTA [pH 8], and 10 mM Tris [pH 8], with protease inhibitor). The chromatin was sonicated for DNA fragmentation and analyzed with agarose gel electrophoresis. The majority of the DNA fragments were in the 500–100 bps range. Cell debris was pelleted by spinning at 10,000 × g for 10 min, and the supernatant was used for chromatin immunoprecipitation (ChIP). 20 μl chromatin supernatant was retained as the DNA input control. Sheep anti-rabbit IgG-conjugated Dynabeads (Dynal) were mixed with antibodies from commercial sources (α-GATA3, Santa Cruz sc-22206; α-MAF, Santa Cruz sc-10017X; α-MYB, Abcam ab45150) in blocking solution (PBS containing 5 mg/ml BSA) o/n at 4°C. Beads were collected and washed three times with PBS/BSA. Chromatin was equally divided into sample and control, adjusted with Triton-X-solution (0.1% Triton X-100, 0.1% sodium deoxycholate, and 1 mM PMSF) and mixed with magnetic beads pre-coated antibodies and incubated at 4°C o/n. Next day, magnetic beads were collected, put on magnetic stand and the supernatant was removed. Then beads were washed with 1 ml RIPA buffer (50 mM HEPES [pH 7.6], 1 mM EDTA, 0.7% sodium deoxycholate, 1% Nonidet P-40, and 0.5 M LiCl, with protease inhibitor mixture) at 4°C to remove nonspecific binding. The beads were collected and washed with RIPA buffer a total of 8-10 times. After washing once with 1 ml TE buffer, the beads were collected by centrifugation at 2000 × g for 2 min and resuspended in 50 μl elution buffer (50 mM Tris [pH 8], 10 mM EDTA, and 1% SDS). Chromatin was eluted from the beads by incubation at 65° for 30 min in a thermo-mixer followed by centrifugation for 1 min at 2000 × g. A total of 50 μl eluted supernatants were removed and mixed with 120 μl TE buffer. Similarly, 20 μl chromatin input control was mixed with 150 μl TE buffer. Decrosslinking was performed by incubation at 65°C overnight. Proteins in the DNA sample were digested in 120 μl proteinase K solution (2% glycogen, 5% proteinase K stock solution, and TE) for 2 h at 37°C. The DNA was then extracted twice with phenol and once with 24:1 chloroform/isoamyl alcohol. The sample was adjusted to 200 mM NaCl. After ethanol precipitation, the DNA was dissolved in 30 μl TE containing 10 μg DNase-free RNase A and incubated for 2 h at 37°C. The DNA was further purified with a Qiagen PCR kit (Qiagen). Libraries for sequencing were generated as previously described in Hawkins et al, Distinct epigenomic landscapes of pluripotent and lineage-committed human cells. Cell Stem Cell 6, 479-491 (2010).

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg38

Number of total reads
35512057
Reads aligned (%)
99.1
Duplicates removed (%)
5.0
Number of peaks
932 (qval < 1E-05)

hg19

Number of total reads
35512057
Reads aligned (%)
98.1
Duplicates removed (%)
5.9
Number of peaks
987 (qval < 1E-05)

Base call quality data from DBCLS SRA