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| Status |
Public on Apr 03, 2014 |
| Title |
KIKO V ER alpha ChIP-seq |
| Sample type |
SRA |
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| Source name |
uterus
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| Organism |
Mus musculus |
| Characteristics |
tissue: uterus
treatment: saline vehicle
antibody: ERα (sc-542, Santa Cruz Inc, Santa Cruz CA)
background mouse strain: B6:129
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| Treatment protocol |
mice were ovariectomized and injected intraperitoneally 10-14 days later with saline vehicle or estradiol (250 ng/mouse). Uterine tissue was collected one hour later
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| Growth protocol |
mice were maintained on NIH-31 chow, standard light/dark cycle, 1-5 mice per cage
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| Extracted molecule |
genomic DNA |
| Extraction 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).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
Sequences (36 nucleotide reads) were aligned to the mouse genome (National Center for Biotechnology Information Build 37.1/mm9) using Eland (Illumina pipeline) software.
Duplicate mapped reads were removed with MarkDuplicates.jar from the Picard suite (v1.62).
ER alpha peak calls were made with Partek Genomics Suite (v 6.11.1101; Partek, Inc, St Louis, MO) using default parameters.
Called peaks were filtered to exclude peaks with Mann-Whitney P-value > 0.01.
Mapped reads were extended at their 3' end to a length equal to the estimated average fragment size for that library and then converted to aggregate coverage (bedGraph format) with BEDtools genomeCoverageBed.
Genome_build: mm9
Supplementary_files_format_and_content: bedGraph file per sample showing aggregate coverage of mapped extended reads; generated by BEDtools genomeCoverageBed.
Supplementary_files_format_and_content: Called ER alpha peaks (idnetified via Partek Genomic Suite) in BED format.
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| Submission date |
Apr 02, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Sylvia C Hewitt |
| E-mail(s) |
sylvia.hewitt@nih.gov, curtiss@niehs.nih.gov
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| Phone |
9842874317
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| Organization name |
NIEHS
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| Department |
RDBL
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| Lab |
Pregnancy & Female Reproduction
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| Street address |
111 Alexander Dr
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| City |
Research Triangle Park |
| State/province |
NC |
| ZIP/Postal code |
27709 |
| Country |
USA |
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| Platform ID |
GPL11002 |
| Series (2) |
| GSE56466 |
ERa and PolII ChIP seq from KIKO mouse uterus |
| GSE56501 |
ER and RNA PolII ChIP-seq in WT and ER mutant mouse uterus |
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| Relations |
| BioSample |
SAMN02716094 |
| SRA |
SRX508006 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1361945_KIKO_Veh_ER.bed.gz |
25.1 Kb |
(ftp)(http) |
BED |
| GSM1361945_KIKO_Veh_ER.fragsize120.bedGraph.gz |
278.8 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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