Antigen

Antigen Class

Input control

Antigen

Input control

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

liver

strain

C57BL/6

genotype

Esr1 wt

hormone treatment

E2

chip antibody

none

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Livers were sliced into small pieces and disrupted in 2 ml PBS through a 21G syringe needle. Cells from one liver were fixed in 10 ml PBS containing 1% formaldehyde for 10 min at room temperature. The cross-linking was next stopped by incubation with glycine at 0.125M final concentration at room temperature under gentle agitation. Cells were recovered by centrifugation at 2,500 x g at 4°C, washed twice with PBS and then lysed in 1 ml of specific buffer [10mM Tris-HCl (pH 8.0), 10mM NaCl, 3 mM MgCl2, 0.5% Igepal]. Extraction of nuclei was performed by applying 40 strokes of potter on the suspension in an ice-cold mortar and further incubation for 5 min at 4°C. Nuclei suspensions originating from each liver were divided into 2 and the membrane debris were eliminated by 2 washes in 2 ml of PBS and centrifugation at 1,000 x g at 4°C. Nuclei were then lysed in 4 ml of nuclei lysis buffer [10 mM EDTA, 50 mM Tris-HCl (pH 8.0), 1% SDS] by incubation on ice during 10 min and three rounds of 20 sec sonication using a Branson 250 apparatus at 50% power. Samples were allowed to cool down 1 min on ice between each sonication pulse. SDS was then neutralized by adding 400 µl of 10% Triton X-100 and chromatin further sonicated into fragments of 150-400 bp mean size by 2 additional 14 min sonications of the lysed nuclei (divided into 12 aliquots of 300 µl) in a BioRuptor apparatus (Diagenode) operating at high intensity with 30 sec on/off duty cycles. Chromatin was then cleared through a 10 min centrifugation at 10,000 x g and the supernatants pooled for further use. ChIP experiments were performed using 300 µl of these chromatin preparations. A tenth of this fraction (30 µl) served to prepare the input DNA, while the rest was diluted 5-fold in IP buffer [2 mM EDTA, 100 mM NaCl, 20 mM Tris-HCl (pH 8.1), and 0.5%Triton X-100] containing 10 μg of yeast tRNA. Immunoprecipitations were performed overnight at 4°C under rocking using 2 μg of antibodies. Complexes were recovered after a 4 hr incubation at 4°C with 5 μg yeast tRNA and 60 μl of 50% protein A or G-Sepharose (Amersham Pharmacia Biosciences) slurry. Precipitates were then serially washed, using 300 μl of Washing Buffers (WB) I [2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 0.1% SDS, 1% Triton X-100, 150 mMNaCl], WB II [2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 0.1% SDS, 1% Triton X-100, 500 mM NaCl], WB III [1 mM EDTA, 10 mM Tris-HCl (pH 8.1), 1% NP-40, 1% Deoxycholate, 0.25 M LiCl] and then twice with 1 mM EDTA, 10 mM Tris-HCl (pH 8.1). Precipitated complexes were removed from the beads through two sequential incubations of the beads with 75 μl of 1% SDS+ 0.1 M NaHCO3 during 15 min with strong agitation. Cross-linking of input and immunoprecipitated samples was reversed by heat at 65°C overnight. DNA was purified on NucleoSpin™ columns (Macherey-Nagel) using NTB buffer. Multiplexed libraries (x3) were prepared according to Illumina's instructions by IGBMC sequencing facility (Strasbourg, France)

Sequencing Platform

instrument_model

Illumina Genome Analyzer II

mm10

Number of total reads

41311778

Reads aligned (%)

98.2

Duplicates removed (%)

40.1

Number of peaks

579 (qval < 1E-05)

mm9

Number of total reads

41311778

Reads aligned (%)

97.9

Duplicates removed (%)

40.0

Number of peaks

620 (qval < 1E-05)