Antigen

Antigen Class

Histone

Antigen

H3K4me3

Cell type

Cell type Class

Liver

Cell type

Hep G2

Primary Tissue

Liver

Tissue Diagnosis

Carcinoma Hepatocellular

Sample

source_name

human hepatocytes (HepG2 H1.3)

tumour stage

transformed

source

liver

antibody

H3K4me3

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Briefly, lysates from cells crosslinked for 10 min in 1% formaldehyde were layered onto a 1.6 M sucrose cushion (1.6 M sucrose in 60 mM, KCl 15 mM NaCl, 5 mM MgCl2, 0.1 mM ethylene glycol tetraacetic acid (EGTA), 15 mM Tris-HCl (pH 7.5), 0.5 mM DTT, 0.1 mM phenylmethanesulfonylfluoride (PMSF), 3.6 ng/mL aprotinin) and centrifuged. Nuclei were collected and portions corresponding to 50μg chromatin were sheared by ultrasonic treatment using a Bioruptor UCD-200 (Diagenode) and 30× [30 s ON/30 s OFF] at position ‘high’. Chromatin fragment size was controlled on agarose gels, and one of the chromatin aliquots was saved as input. For ChIP analyses, sheared chromatin samples were incubated with the respective antibody-of-interest in a rotator for 16 h at 4°C. Subsequently 20 μl protein G magnetic beads (Diagenode) were added and incubated for 4 hrs at 4°C rotating. Immunocomplexes attached to protein G magnetic beads were separated using a magnetic rack and washed repeatedly. To elute DNA fragments enriched by immunoprecipitation, immunocomplexes were incubated with elution buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl, (pH 8.1)) for 30 min at 65°C on a shaker. Eluted immunocomplexes were treated with proteinase K overnight at 55°C. Antibodies used for IP were directed against HBxAg (mouse monoclonal antibody, clone 3F6-G10; Bio-Rad Laboratories Inc.), Pol2 (Active Motif, Carlsbad, CA, USA), H3K4me3 (Diagenode, Liège, Belgium), H3K4me1 (Diagenode, Liège, Belgium), H3K9me3 (Diagenode, Liège, Belgium), H3K9ac (Diagenode, Liège, Belgium), H3K27me3 (Diagenode, Liège, Belgium), H3K27ac (Diagenode, Liège, Belgium), H3K36me3 (Diagenode, Liège, Belgium) or H3K36ac (Diagenode, Liège, Belgium). DNA associated with enriched immunocomplexes was purified by phenol:chloroform:isoamylic alcohol extraction and precipitated with isopropylic alcohol. Illumina-compatible libraries were prepared using the MicroPlex Library Preparation kit v2 (Diagenode, Liège, Belgium). Massive parallel sequencing was performed as described below. ChIP-seq was performed with the paired-end or, alternatively, with the single read option (42nt paired-end in mmu experiments/50nt single-end in hsa experiments) and the Illumina NextSeq 500 High Output Kit

Sequencing Platform

instrument_model

NextSeq 500

hg38

Number of total reads

29213940

Reads aligned (%)

98.9

Duplicates removed (%)

3.0

Number of peaks

987 (qval < 1E-05)

hg19

Number of total reads

29213940

Reads aligned (%)

98.2

Duplicates removed (%)

4.2

Number of peaks

980 (qval < 1E-05)