Antigen

Antigen Class

Input control

Antigen

Input control

Cell type

Cell type Class

Blood

Cell type

Thymocytes

MeSH Description

HEMATOPOIETIC PROGENITOR CELLS that have migrated to the THYMUS where they differentiate into T-LYMPHOCYTES. Thymocytes are classified into maturational stages based on the expression of CELL SURFACE ANTIGENS.

Sample

source_name

Thymocytes

cell type

TCR-int CD69-pos thymocyte

strain

C57BL/6

genotype

OT-II

chip antibody

none

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Thymi were harvested, processed for single cell suspension, and stained with antibody for cell sorting by FACS Aria. Sorted samples were cross-linked with 1% formaldehyde (final concentration) for 10 min and quenched with 125 mM glycine for 5 min at room temperature. After washing with TBS, cells were resuspended in cell lysis buffer (10 mM Tris-HCl, pH7.5, 10 mM NaCl, 0.5% NP-40) and incubated on ice for 10 min. Lysates were washed with MNase digestion buffer (20 mM Tris-HCl, pH7.5, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2). After resuspendsion in 200 μL fresh MNase digestion buffer containing a proteinase inhibitor cocktail (Sigma), the lysates were incubated with 2,000 units of MNase (NEB, Cat # M0247S) per 4 x 106 cells at 37 °C for 20 min with continuous mixing in thermal mixer. After adding 200 μL of sonication buffer (100 mM Tris-HCl, pH8.1, 20 mM EDTA, 200 mM NaCl, 2% Triton X-100, 0.2% sodium deoxycholate), the lysates were sonicated for 30 min (30 sec on / 30 sec off) using Bioruptor Twin (UCD-400) (Diagenode, Inc.) and centrifuged at 21,130 x g for 10 min. The supernants were collected, and the chromatin content was estimated by the Qubit assay (Invitrogen). For normalization of ChIP efficiency, Drosophila chromatin equivalent to about 5% of total chromatin was added. The chromatin was then incubated with anti-H3K27ac antibody (Abcam, Cat.# ab4729, lot# GR150367) or in-house generated anti-H3K9ac antibody (EDL lot 1) on a rocker overnight. Protein G-magnetic beads (30 μL, Life Technologies) were added, and further incubated for 3 hours. The beads were extensively washed with ChIP buffer (50 mM Tris-HCl, pH8.1, 10 mM EDTA, 100 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate), high salt buffer (50 mM Tris-HCl, pH8.1, 10 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate), LiCl2 buffer (10 mM Tris-HCl, pH8.0, 0.25 M LiCl2, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA), and TE buffer. Bound chromatin was eluted and reverse-crosslinked at 65°C overnight. DNAs were purified using Min-Elute PCR purification kit (Qiagen) after the treatment of RNase A and proteinase K. ChIP-seq libraries were prepared from about 5 ng ChIP and input DNA using the ThruPLEX® DNA-seq Kit V2 (Rubicon Genomics, Ann Arbor, MI) according to manufacturere protocol.

Sequencing Platform

instrument_model

Illumina HiSeq 4000

mm10

Number of total reads

31276532

Reads aligned (%)

91.3

Duplicates removed (%)

5.2

Number of peaks

263 (qval < 1E-05)

mm9

Number of total reads

31276532

Reads aligned (%)

91.2

Duplicates removed (%)

5.4

Number of peaks

232 (qval < 1E-05)