Antigen

Antigen Class

Histone

Antigen

H3K4me3

Cell type

Cell type Class

Liver

Cell type

Liver

MeSH Description

A large lobed glandular organ in the abdomen of vertebrates that is responsible for detoxification, metabolism, synthesis and storage of various substances.

Sample

source_name

Rat liver

chip antibody

H3K4me3 (Active Motif, 39915)

strain

Sprague-Dawley

treatment

Sesame oil (VEH)

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Frozen liver tissue was pulverized, followed by cross-linking of proteins to DNA with 37% formaldehyde, and then incubation with 10X glycine to stop the reaction. Cross-linked tissue was homogenized, and centrifuged at 1,500 rpm for 5 min at 4°C. The cell pellet was resuspended with cell lysis buffer (PBS with 0.5 mM EDTA and 0.05% Triton X-100), incubated on ice for 15 min, followed by centrifiugation at 5,000 rpm for 5 min at 4°C. The cell pellet was then resuspended with nuclear lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl at pH 8.1) and incubated on ice for 10 min. Chromatin was sonicated with a bioruptor (Diagenode, Denville, NJ) to obtain fragment sizes of 100-300 bp for ChIP-seq. ChIP was performed by incubating sheared chromatin with Magna ChIPTM Protein A+G magnetic beads, and antibodies against H3K4me3, H3K4me1, H3K27ac or H3K27me3 overnight at 4°.The next day, the beads were washed with low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8, and 150 mM NaCl) high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8, and 500 mM NaCl), lithium chloride wash buffer (0.25 M LiCl, 1% IGEPAL CA630, 1% sodium deoxycholate, 1 mM EDTA, and 10 mM Tris-HCl pH 8), and TE buffer (20 mM Tris-HCl pH 8 and 1 mM EDTA) for 5 minutes each at 4°. Immunoprecipitated DNA was recovered from the beads by incubation with ChIP elution buffer (1% SDS, 1 mM EDTA, 50 mM NaHCO3, and 50 mM Tris-HCl pH 8) and proteinase K for 2 hours at 62° with shaking, followed by incubation at 95° for 10 minutes. DNA was purified using the QIAquick PCR Purification kit (Qiagen) and DNA concetration was measured using a NanoDrop spectrophotometer (Thermo Scientific). Sequencing librairies were prepared using Bioo Kit Option 2 protocol using 1.7-10 ng of DNA per sample.

Sequencing Platform

instrument_model

Illumina HiSeq 2500

rn6

Number of total reads

30200147

Reads aligned (%)

71.9

Duplicates removed (%)

32.2

Number of peaks

19271 (qval < 1E-05)