Antigen

Antigen Class

Histone

Antigen

H3K27ac

Cell type

Cell type Class

Digestive tract

Cell type

Ileum

MeSH Description

The distal and narrowest portion of the SMALL INTESTINE, between the JEJUNUM and the ILEOCECAL VALVE of the LARGE INTESTINE.

Sample

source_name

ileum epithelial cells

genotype

C57BL/6

diet

Chow diet

chip antibody

H3K27ac, Abcam ab4729

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Mouse ileum was washed with ice-cold PBS and IECs were extracted in 10 mM EDTA. Cells were washed with ice-cold PBS twice and fixed in 1% formaldehyde at room temperature for 10 min. Cells were then quenched in 125 mM glycine for 10 min, washed twice with PBS, resuspended in 0.5 ml lysis buffer (20 mM Tris-HCl, pH 8, 60 mM KCl, 1 mM EDTA, 0.5% NP-40 with protease inhibitors), and incubated at 4oC for 15 min. Nuclei were pelleted and resuspended in 250 μl RIPA buffer (Thermo Scientific 89900) with protease inhibitors and sonicated with a Bioruptor Pico sonication device (Diagenode) for 20 cycles (30 sec on, 30 sec off). The supernatant was pre-cleared with 10 μl protein G magnetic beads for 1 h and then incubated with 3 μg of primary antibodies overnight at 4oC. The antibodies used were: anti-H3K9ac (Abcam ab4441), anti-H3K27ac (Abcam ab4729), anti-HDAC3 (Abcam ab7030), anti-PGC-1α (Millipore, ST1204), anti-ERRα (Millipore 17-603). 50 μl protein G beads were added and incubated for 1.5 h at 4oC. Beads were washed four times with LiCl wash buffer (100 mM Tris-HCl pH 7.5, 500 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate) and finally with TE buffer. DNA was eluted in 150 μl TES buffer (TE with 1% SDS, 150 mM NaCl, and 5 mM dithiothreitol) by resuspending the beads at 65oC for 8 h. DNA was purified using ChIP DNA Clean & Concentrator (Zymo Research D5205).  ChIP-seq libraries were generated using a KAPA Hyper Prep Kit (kapabiosystems KK8502) and library sizes and concentrations were evaluated by Bioanalyzer and RT-qPCR.

Sequencing Platform

instrument_model

Illumina HiSeq 2500

mm10

Number of total reads

45851404

Reads aligned (%)

98.5

Duplicates removed (%)

18.8

Number of peaks

12999 (qval < 1E-05)

mm9

Number of total reads

45851404

Reads aligned (%)

98.3

Duplicates removed (%)

18.8

Number of peaks

12995 (qval < 1E-05)