Antigen

Antigen Class

Input control

Antigen

Input control

Cell type

Cell type Class

Blood

Cell type

Th1 Cells

MeSH Description

A subset of helper-inducer T-lymphocytes which synthesize and secrete INTERLEUKIN-2; INTERFERON-GAMMA; and INTERLEUKIN-12. Due to their ability to kill antigen-presenting cells and their lymphokine-mediated effector activity, Th1 cells are associated with vigorous delayed-type hypersensitivity reactions.

Sample

source_name

In vitro differentiated Th1 cells

cell type

Th1

antibody

input

treatment/agent

anti-CD3/CD28

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Cells 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 detergent. 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 pre-incubated with 20 µl of anti-T-bet 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 RNase A, proteinase K and phenol:chloroform extraction followed by ethanol precipitation. 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. The libraries were quantified using a Qubit and Agilent bioanalyzer, pooled and subjected to 150 bp single-end read sequencing with an Illumina NextSeq sequencer.

Sequencing Platform

instrument_model

NextSeq 500

hg38

Number of total reads

160899469

Reads aligned (%)

94.3

Duplicates removed (%)

10.2

Number of peaks

23976 (qval < 1E-05)

hg19

Number of total reads

160899469

Reads aligned (%)

93.7

Duplicates removed (%)

11.4

Number of peaks

22439 (qval < 1E-05)