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| Status |
Public on Jul 01, 2015 |
| Title |
ChIP ESR1 preantral follicles |
| Sample type |
SRA |
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| Source name |
cells from preantral follicles maintained for 9 days in vitro and treated for 45 minutes with 10-7M estradiol
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| Organism |
Mus musculus |
| Characteristics |
strain: SWISS
Sex: female
tissue: ovary
age: 8-week
antibody: anti-ESR1 C-terminal antibody (Santa Cruz sc543)
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| Treatment protocol |
45 minutes before fixation, cells were treated with 100nmol/L 17beta-estradiol (Sigma-Aldrich)
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| Growth protocol |
The ovaries of 20 8-week old female Swiss mice were dissected and digested in DMEM/F-12 medium (Gibco) containing 1mg/mL collagenase (Sigma-Aldrich), 0,5mg/mL BSA (Sidma-Aldrich) and 2000 U/mL DNase I (Thermo-Fisher), in a ratio of 500µL per ovary, at 37°C for 30 min. Ovaries were progressively dispersed during digestion using pipette tips of decreasing size to flush the organ debris. The mix was then diluted in 10 volumes of DMEM/F-12 medium, filtered using a 500 µm membrane, spinned at 300 g. The pellet was rinsed in cold PBS and spinned again, and the pelleted debris were resuspended in a PBS/Ficoll mix of density 1.10. A PBS/Ficoll gradient was made by depositing PBS/Ficoll layers of density 1.08, 1.07, 1.06, 1.05, 1.03 and 1. The gradient was spinned at 1500 g for 20min, and 1 mL fractions were collected and diluted in 5 mL culture medium (i.e. DMEM/F-12 supplemented with 4% FBS, 1% Insulin-Transferrin-Selenium-X supplement, 1% Penicillin-Streptomycine (Gibco) and 10-7M androstenedione (Sigma-Aldrich). We then manually collected intact preantral follicles, characterized by the presence of several layers of granulosa cells around the oocyte and the absence of antrum. They mainly migrated to the 1.07/1.06 and 1.06/1.05 interfaces. Follicles were then rinsed, transferred to culture plates and grown overnight. On day 1, they were dispersed using trypsin and then cultured in monolayers. Culture medium was changed on day 3, and cells were passed on days 5 and 7.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells grown to confluence in three 150-mm plates were fixed in situ with 1% Formaldehyde for 10 min at room temperature, fixation was then blocked with 125 mM glycine, cells were rinsed and scrapped in PBS. Cells were first lysed in Lysis buffer A (50 mM Hepes pH 7.5, NaCl 140 mM, EDTA 1 mM, glycerol 10% NP-40 0.5% and Triton X-100 0.25%), then spinned and rinsed in lysis buffer B (Tris 10 mM pH 7.5, NaCl 200 mM, EDTA 1 mM, EGTA 0.5 mM), centrifugated again and finally resuspended in lysis buffer C (Tris 10 mM pH 7.5, NaCl 100 mM, EDTA 1 mM, EGTA 0.5 mM, Na–deoxycholate 0.5%, N-lauryl sarcosine 0.5%). Chromatin was sheared in a Bioruptor sonicator, used at high power with 30/30 s cycles for 20 min, then 1% Triton X-100 was added to lysates, and cell debris were centrifugated. In parallel, 30 μg anti-FOXL2 or anti-ESR1 were incubated with 200 μl ProteinG Dynabeads for 4 h in PBS containing 0.1% BSA. Lysates were incubated with the beads overnight, beads were then washed five times in wash buffer (50 mM Hepes pH 7.6, 500 mM LiCl, 1 mM EDTA, 0.7% Na–deoxycholate, 1% NP-40) and once in TBS. DNA–protein complexes were then eluted in 10 mM Tris pH 8, 1 mM EDTA, 1% SDS at 65 °C for 15 min. Crosslinks were reversed at 65 °C overnight, then recovered DNA was treated with RNaseA, proteinase K and purified using phenol–chloroform. Input samples underwent to the same procedure.
Adapters were ligated using a SPRI-TE Nucleic Acid Extractor Robot with a SPRIworks Fragment Library Kit I and adapters from TruSeq DNA LT Sample Prep kit (Illumina). Ligated fragments were amplified with C and D primers(C primer :5’-AATGATACGGCGACCACCGAGATCTACAC-3’b and D primer : 5’-CAAGCAGAAGACGGCATACGAGAT-3’) using Phusion HF polymerase (Thermo Fisher Scientific) for 14 PCR cycles. 200 to 400 bp fragments were selected on gel and purified on a Qiagen column.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 1000 |
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| Description |
DNA ChiPed with anti-ESR1 C-terminal antibody (Santa Cruz sc543)
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| Data processing |
Base calling, CASAVA-1.8.2
Adapter removal, cutadapt-1.2.1
Fastq conversion, Fastqc-0.10.1
Read alignment, Bowtie Galaxy version v1.1.2, Full list of parameters : Will you select a reference genome from your history or use a built-in index?:indexed;Select a reference genome:mm9female;Is this library mate-paired?:single;FASTQ file:1: Foxl2;Bowtie settings to use:full;Skip the first n reads (-s):0;Only align the first n reads (-u):-1;Trim n bases from high-quality (left) end of each read before alignment (-5):0;Trim n bases from low-quality (right) end of each read before alignment (-3):0;Maximum number of mismatches permitted in the seed (-n):2;Maximum permitted total of quality values at mismatched read positions (-e):70;Seed length (-l):28;Whether or not to round to the nearest 10 and saturating at 30 (--nomaqround):Round to nearest 10;Number of mismatches for SOAP-like alignment policy (-v):-1;Whether or not to try as hard as possible to find valid alignments when they exist (-y):Do not try hard;Report up to n valid alignments per read (-k):1;Whether or not to report all valid alignments per read (-a):Do not report all valid alignments;Suppress all alignments for a read if more than n reportable alignments exist (-m):1;Write all reads with a number of valid alignments exceeding the limit set with the -m option to a file (--max):False;Write all reads that could not be aligned to a file (--un):False;Whether or not to make Bowtie guarantee that reported singleton alignments are 'best' in terms of stratum and in terms of the quality values at the mismatched positions (--best):noBest;Maximum number of backtracks permitted when aligning a read (--maxbts):125;Override the offrate of the index to n (-o):-1;Seed for pseudo-random number generator (--seed):-1;Suppress the header in the output SAM file:False;
Unmapped reads removal, Filter SAM galaxy version 1.0.0
Peak calling, MACS Galaxy version 1.0.1, Full list of parameters : Tag size:50;Band width:160;Pvalue cutoff for peak detection:0,00001;Select the regions with MFOLD high-confidence enrichment ratio against background to build model:20;Parse xls files into into distinct interval files:False;Save shifted raw tag count at every bp into a wiggle file:wig;Extend tag from its middle point to a wigextend size fragment.:-1;Resolution for saving wiggle files:20;Use fixed background lambda as local lambda for every peak region:False;3 levels of regions around the peak region to calculate the maximum lambda as local lambda:1000,5000,10000;Build Model:create_model;Diagnosis report:no_diag;Perform the new peak detection method (futurefdr):True;
Genome_build: mm9
Supplementary_files_format_and_content: .bed file contain 5 columns : chr, peakStart, peakEnd, peakID and score
Supplementary_files_format_and_content: .wig file contain unnormalized read mapping abundancies (20bp digits)
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| Submission date |
Oct 21, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Adrien Georges |
| Organization name |
CNRS
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| Department |
FRE3377
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| Lab |
Equipe WERNER/SOUTOURINA
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| Street address |
CEA/SACLAY
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| City |
GIF SUR YVETTE |
| ZIP/Postal code |
91190 |
| Country |
France |
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| Platform ID |
GPL15103 |
| Series (1) |
| GSE62545 |
FOXL2 and ESR1 binding sites in primary cells from mouse ovarian follicles |
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| Relations |
| BioSample |
SAMN03121687 |
| SRA |
SRX737011 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1528688_ChIP-ESR1.bed.gz |
134.4 Kb |
(ftp)(http) |
BED |
| GSM1528688_ChiP-ESR1.wig.gz |
127.3 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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