Sample information curated by ChIP-Atlas

Antigen

Antigen Class
RNA polymerase
Antigen
RNA polymerase II

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
In vitro differentiated Th2 cells
cell type
Th2
antibody
Santa-Cruz N-20 (sc-899)
treatment/agent
unstimulated

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Human Th1 and Th2 cells, pooled from multiple donors, were crosslinked by the addition of one-tenth volume of fresh 11% formaldehyde solution for 20 minutes at room temperature before the reaction was quenched by addition of glycine. Cells were rinsed twice with 1xPBS and flash frozen in liquid nitrogen. Cells were lysed with non-ionic detergent, nuclei washed and then lysed with ionic detegerent. Cells were sonicated on ice to solubilize and shear crosslinked DNA (24W for 10 x 30 second pulses using a Misonix Sonicator 3000). The resulting whole cell extract was cleared by centrifugation and then incubated overnight at 4°C with 100 µl of Dynal Protein G magnetic beads that had been preincubated with 10 ug of purified antibody or, for the case of T-bet, 10 ul of purified serum. Beads were washed 6 times with RIPA buffer and 1 time with TE containing 50 mM NaCl. Bound complexes were eluted from the beads by heating at 65°C with occasional vortexing and crosslinks then reversed in IP and input DNA by overnight incubation at 65°C. IP and input DNA were then purified by treatment with RNAseA, proteinase K and phenol:chloroform extraction followed by ethanol precipitation. H3K4me3 ChIP was performed on native chromatin derived from ex vivo differentiated human Th1 and Th2 cells. Chromatin was prepared broadly using the protocol of Feil and colleagues with some minor modifications (http://www.epigenome-noe.net/researchtools/protocol.php?protid=2). Mono and dinucleosomal chromatin was recovered from nuclei treated with micrococcal nuclease (10U/µl for 7 mins). Chromatin quality was assessed by agarose gel elecrophoresis and semi-quantitated using a nanodrop. A second chromatin prep was also prepared from Th1 and Th2 cells, these cells were exposed to fomaldehyde in order to cross link protein to DNA. Nuclei from these cells were treated with MNase followed by sonication (6x20 second bursts at 40% amplitude using a Sonics VibraCell probe sonicator). ChIP for H3K4me3 and total H3 was performed with Protein G beads (Active Motif). DNA was purified by phenol/chloroform phase separation, ethanol precipitation, followed by Qiagen clean up. Libraries were constructed from ChIP and input DNA by standard Illumina protocols, except that DNA in the range 150-350bp was gel-purified after PCR-amplification.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg38

Number of total reads
22824455
Reads aligned (%)
70.3
Duplicates removed (%)
7.2
Number of peaks
26610 (qval < 1E-05)

hg19

Number of total reads
22824455
Reads aligned (%)
69.9
Duplicates removed (%)
7.9
Number of peaks
26626 (qval < 1E-05)

Base call quality data from DBCLS SRA