Antigen

Antigen Class

TFs and others

Antigen

Esr1

Cell type

Cell type Class

Uterus

Cell type

Uterus

MeSH Description

The hollow thick-walled muscular organ in the female PELVIS. It consists of the fundus (the body) which is the site of EMBRYO IMPLANTATION and FETAL DEVELOPMENT. Beyond the isthmus at the perineal end of fundus, is CERVIX UTERI (the neck) opening into VAGINA. Beyond the isthmi at the upper abdominal end of fundus, are the FALLOPIAN TUBES.

Sample

source_name

uterus

tissue

uterus

treatment

estradiol (250 ng/mouse)

antibody

ERα (sc-542, Santa Cruz Inc, Santa Cruz CA)

background mouse strain

B6:129

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Uteri were submersed in PBS + 1% formaldehyde, cut into small (~1 mm3) pieces with a razor blade and incubated at room temperature for 15 minutes. Fixation was stopped by the addition of 0.125 M glycine (final). The tissue pieces were then treated with a TissueTearor (Biospec Products, Bartlesville, OK) and finally spun down and washed 2x in PBS. Chromatin was isolated from the sample by adding 5-10 ml lysis buffer containing PIPES, Igepal, PMSF and Protease Inhibitor Cocktail, followed by disruption with a Dounce homogenizer. Samples were pelleted by centrifugation and resuspended in buffer containing Na deoxycholate, SDS, and Triton X-100. Lysates were sonicated using a Misonix Sonicator 3000 (Misonix, Farmingdale, NY) equipped with a microtip in order to shear the DNA to an average length of 300-500 bp. Lysates were cleared by centrifugation and the chromatin suspensions were transferred to new tubes and stored at -80 C. To prepare Input DNA (genomic DNA), two aliquots of 10-25 µl each (approximately 1/50 of each chromatin preparation) were removed and treated with RNase for 1-2 h at 37 C, proteinase K for 3 h at 37 C, and 65 C heat for at least 6 h to overnight for de-crosslinking. DNAs were purified by phenol-chloroform extraction and ethanol precipitated. Pellets were resuspended in 1/5 TE. Resulting DNAs were quantified on a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE). Extrapolation to the original chromatin volume allowed determination of the yield for each chromatin preparation, as measured by the DNA content. Prior to use in ChIP, protein A agarose beads (Invitrogen Technologies, Carlsbad, CA) were preblocked using blocking proteins and nucleic acids for 3 h. For each ChIP reaction, an aliquot of chromatin (20-30 µg) was precleared with 30 µl preblocked protein A agarose beads for 1-2 h. ChIP reactions were set up using precleared chromatin and antibody to ERα (sc-542, Santa Cruz Inc, Santa Cruz CA) or PolII phospho Ser5 (ab5095, Abcam, Cambridge, MA) in a buffer containing Na deoxycholate and incubated overnight at 4 C. Preblocked protein A agarose beads were added and incubation at 4 C was continued for another 3 h. Agarose beads containing the immune complexes were washed two times each with a series of buffers consisting of the deoxycholate sonication buffer, high salt buffer, LiCl buffer, and TE buffer. An SDS-containing buffer was added to elute the immune complexes from the beads, and the eluates were subjected to RNase treatment at 37 C for 20 min and proteinase K treatment at 37 C for 3 h. Crosslinks were reversed by incubation overnight at 65 C, and ChIP DNAs were purified by phenol/chloroform extraction and ethanol precipitation. Quality of ERα ChIP enrichment was assayed by qPCR using primers against candidate EREs in the Igf1 and Stat5a promoters (9). Quality of PolII ChIP enrichment was assayed by qPCR using primers against region in Intron 1 of the housekeeping genes Actin B and Gapdh. Input DNA was queried at the same sites in parallel. ChIP DNA was amplified by following the Illumina ChIP-Seq DNA Sample Prep Kit (Illumina, Inc. San Diego, California) protocol. In brief, DNA ends were polished and 5’-phosphorylated using T4 DNA polymerase, Klenow polymerase and T4 polynucleotide kinase. After addition of 3’-A to the ends using Klenow fragment (3’-5’ exo minus), Illumina genomic adapters were ligated and the sample was size fractionated (200-250 bp) on a 2% agarose gel. After a final PCR amplification step (18 cycles, Phusion polymerase, New England Bioloabs, Ipswich, MA), the resulting DNA libraries were quantified and tested by qPCR at the same specific genomic regions as the original ChIP DNA to assess quality of the amplification reactions. DNA libraries were sequenced on a Genome Analyzer II (Illumina).

Sequencing Platform

instrument_model

Illumina Genome Analyzer IIx

mm10

Number of total reads

48508817

Reads aligned (%)

86.5

Duplicates removed (%)

23.3

Number of peaks

41494 (qval < 1E-05)

mm9

Number of total reads

48508817

Reads aligned (%)

86.5

Duplicates removed (%)

23.4

Number of peaks

41474 (qval < 1E-05)