Antigen

Antigen Class

No description

Antigen

NA

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

ENA-CHECKLIST

ERC000011

ENA-FIRST-PUBLIC

2016-01-11T17:01:44Z

ENA-LAST-UPDATE

2018-03-09T01:10:04Z

External Id

SAMEA3403293

INSDC center name

SickKids Research Institute Genetics & Genome Biology Program and Department of Molecular Genetics, University of Toronto

INSDC first public

2016-01-11T17:01:44Z

INSDC last update

2018-03-09T01:10:04Z

INSDC status

public

Submitter Id

E-MTAB-3587:do71

age

2 to 4 month

antibody part number

sc13059

broker name

ArrayExpress

common name

house mouse

individual

C57/AJ_5

method

ChIP-seq

organism part

liver

sample name

E-MTAB-3587:do71

scientific_name

Mus musculus

sex

male

strain

C57BL/6J x A/J

Sequenced DNA Library

library_name

do71

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Samples were prepared as previously described (doi:10.1016/j.ymeth.2009.03.001). Liver material was crosslinked in 1% formaldehyde for 20 minutes followed by a 10 minute incubation in 500 mM glycine buffer to neutralize the formaldehyde. Samples were rinsed with PBS and frozen at -80 Celsius until use. Chromatin immunoprecipitation was performed as previously described (doi:10.1016/j.ymeth.2009.03.001). Chromatin from a mass equivalent of at least 1/4 mouse liver was used for each ChIP experiment. Crosslinked tissues were dounce homogenized into a single cell suspension and rinsed with ice-cold PBS. Each pellet of crosslinked material was resuspended in 10 ml of LB1 (50 mM Hepes-KOH, pH 7.5; 140 mM NaCl; 1mM EDTA; 10% Glycerol; 0.5% NP-40 or Igepal CA-630; 0.25% Triton X-100), rocked at 4 degrees C for 10 minutes, and pelleted by centrifugation at 2,000 x rcf for 4 minutes at 4 degrees C. Pellets were then resuspended in 10 ml of LB2 (10 mM Tris-HCL, pH8.0; 200 mM NaCl; 1 mM EDTA; 0.5 mM EGTA), rocked gently at 4 degrees C for 5 minutes, and pelleted again by centrifugation at 2,000 x rcf for 4 minutes at 4 degrees C. Finally each pellet was resuspended in 3 ml of LB3 (10 mM Tris-HCl, pH 8; 100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 0.1% Na-Deoxycholate; 0.5% N-lauroylsarcosine). Chromatin from the nuclei fraction was fragmented to an average length of 300 bp by sonication. Sonicated lysates were treated with 150uL of 20% triton X-100, and sonication debris was removed by centrifugation at 20,000 x rcf for 10 minutes at 4 degrees C. 150uL of whole cell extract (WCE) was taken as the input control. 100uL of magnetic protein-G Dynabeads (LifeTechnologies, cat. no. 10004D) was washed three times and pre-blocked with 0.5% BSA (Sigma, cat. no. A8412) w/v in PBS. 5-10 ug of antibody was incubated with pre-blocked Dynabeads for at least 6 hours, rotating at 4 degrees C, then washed three times with blocking solution to remove excess unbound antibody. Antibodies used were: Rad21 (Abcam, ab992), Top2b (Santa Cruz Biotech, sc13059), H3K4me3 (Abcam, ab8580), H3K36me3 (clone 13C9 from Hitoshi Kimura), Ctcf (Millipore, mp07-729), H3K4me2 (Millipore, mp07-030). Lysates were added to protein-G bound antibodies and incubated overnight at 4 degrees C. ChIP libraries were made as previously described protocol (doi:10.1016/j.ymeth.2009.03.001). 50 µl of cell lysate from each sonication was removed as the input control DNA that was processed. Libraries were prepared following the instructions of Illumina (Sample preparation for genomic DNA - version 2.2) with the following modifications: The ChIP-enriched DNA and input DNA were not further fragmented. After end-repair and addition of an 'A' base to the 3' ends, the adapters were ligated to the ends of the DNA fragments using 2 µl of forty-fold diluted 'Adapter oligo mix'’ in a total reaction volume of 25 µl. Between these steps, the DNA was purified using the DNA Clean-and-Concentrator-5 kit (Zymo Research). Subsequently, the DNA was amplified by 18 cycles of PCR, purified with QIAquick PCR purification Kit, and eluted with 33.5 µl of 10 mM Tris buffer at pH7.0. The PCR product was sized selected on 2% agarose gel and a gel slice containing the 200-300 bp fragments was excised.

Sequencing Platform

instrument_model

Illumina HiSeq 2500

mm10

Number of total reads

31544741

Reads aligned (%)

93.5

Duplicates removed (%)

24.9

Number of peaks

58478 (qval < 1E-05)

mm9

Number of total reads

31544741

Reads aligned (%)

93.2

Duplicates removed (%)

24.9

Number of peaks

58484 (qval < 1E-05)