Antigen

Antigen Class

TFs and others

Antigen

Junb

Cell type

Cell type Class

Blood

Cell type

Th17 Cells

MeSH Description

A subset of helper-effector T-lymphocytes which synthesize and secrete INTERLEUKINS IL-17; IL-17F; and IL-22. These cytokines are involved in host defenses and tissue inflammation in autoimmune diseases.

Sample

source_name

in vitro-generated TH17 cells

cell type

TH17(beta)

genotype

JUNBFl/Fl

strain

C57BL/6

chip antibody

JUNB

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

ChIP was performed using a SimpleChIP Plus Enzymatic Chromatin IP Kit (Cell signaling) with some modifications. Briefly, activated T cells (10^6/ChIP-Seq or 1-3 x 10^5/ChIP-PCR) were cross-linked in culture medium containing 1% formaldehyde at room temperature for 10 min, and the reaction was stopped by adding glycine solution. THen cells were lysed, and nuclei were collected and treated with micrococcal nuclease (0.0125 μl/ml) for 20 min at 37°C. After stopping the reaction by adding 0.05M EGTA, samples were sonicated with several pulses to disrupt nuclear membranes, and then the supernatant containing chromatin was collected after centrifugation. Chromatin solutions were incubated with 2 μg of antibodies overnight at 4°C with rotation, followed by incubation with Protein G magnetic beads for 2 hr at 4°C. Beads were washed, and the chromatin was eluted. Cross-links were reverted according to kit instructions. Libraries were prepared using KAPA Hyper Prep Kit (KAPA Biosystems) protocols for blunt-ending, polyA extension, and adapter ligation. Post ligation clean-up by Agencort AMPure XP (Beckman Coulter) was performed at a 1.8X DNA ratio to purify ligated DNA, which was then PCR-amplified and purified using AMPure XP at a 1.2X DNA ratio to remove excess adapter-dimer and to preserve small fragments. Size selection was performed with a 2% agarose gel cassette of Blue Pippin (Sage Science) for target insert size between 30 and 180bp. Library quantification was performed by droplet digital PCR (BioRad). All libraries were pooled and loaded on to cBot (50 µL of 200 pM) for cluster generation, and then sequenced on an Illumina HiSeq4000 at a target sequencing depth of 10 million uniquely aligned reads.

Sequencing Platform

instrument_model

Illumina HiSeq 4000

mm10

Number of total reads

11142688

Reads aligned (%)

0.0

Duplicates removed (%)

17.0

Number of peaks

0 (qval < 1E-05)

mm9

Number of total reads

11142688

Reads aligned (%)

0.0

Duplicates removed (%)

17.1

Number of peaks

0 (qval < 1E-05)