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| Status |
Public on Jan 01, 2014 |
| Title |
INPUT_PR_PR_infected+Prog |
| Sample type |
SRA |
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| Source name |
primary HUVECs
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| Organism |
Homo sapiens |
| Characteristics |
cell type: endothelial cells
passage nr.: 4-5
treatment: PR infected + Progesterone
ChIP: none, input
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| Treatment protocol |
Primary HUVECS were infected at passage 4 with a lentivirus containing human PR, then split onto 10cm dishes and grown to confluence (hence they become passage number 5 for the experiment). 3 days after they reached confluence they were treated with progesterone (100nM) for 1 hour--then harvested for the experiment.
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| Growth protocol |
Human umbilical vein endothelial cells, passages 4-6, were cultured in MCDB-131 (VEC Technologies, Rensselaer, NY) with the addition of 10% fetal bovine serum (Omega Scientific, Tarzana, CA) that was stripped using 0.25% dextran coated charcoal (Sigma, St. Louis, MO).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were trypsinized, washed with PBS and crosslinked with 1% formaldehyde in PBS for 10 min at RT. After PBS washing, cells were resuspended in 400 μl of lysis buffer (1% SDS, 20 mM EDTA and 50 mM Tris-HCl (pH 8.0)) containing protease inhibitors (Roche, Indianapolis, IN), incubated for 10 min on ice and sonicated using Misonix cup-horn sonicator to achieve, on average, 200bp fragments for ChIP-seq and 500bp fragments for ChIP-PCR. The lysate was diluted 10 times with ChIP dilution buffer containing 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA and 16.7 mM Tris-HCl (pH 8.1) and immunoprecipitatied with 3 ug of anti-PR or IgG antibody overnight at 4 degrees. 20 μl of the lysates were used as input. The complexes were captured using protein A Dynabeads (Invitrogen, Grand Island, NY) and washed twice with the following buffers: low-salt immune complex wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1); high-salt immune complex wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1) and 500 mM NaCl); LiCl wash buffer (0.25 M LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA and 10 mM Tris-HCl (pH 8.1)) and TE (10 mM Tris-HCl and 1 mM EDTA (pH 8.0)). After elution with 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 1% SDS, crosslinks were reversed by overnight incubation at 65°C. Samples were then treated with RNase A for 30 min at 37°C and proteinase K for 2 h at 56°C. DNA was subsequently purified using Qiagen MinElute Columns according to manufacturers instructions. DNA concentration was measured using a Qubit (Invitrogen, Grand Island, NY).
The library for sequencing was constructed using Ovation Ultralow IL Multiplex System 1-8 according to manufacturer's instructions (Nugen, San Carlos CA). Libraries were sequenced using HIseq-2000 (Illumina, San Diego, CA) to obtain 50 bp long reads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
INPUT
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| Data processing |
Debarcoding of the multiplex runs was performed using Fastx toolkit
Tags were mapped to the human genome (hg19) using bowtie v0.12.7 with parameters -v 2 -m 1 -S --best --strata
Peak identification was performed with MACS v1.3.7.1 using default parameters
Peaks for PR and PR+P conditions were called using either input, negative control or IgG control as a reference and only peaks that were present in all three analyses (minus IgG peaks) were included in the final list of PR binding sites
PR peaks were mapped to nearby genes within 50kb range from TSS using Genomic Regions Enrichment of Annotations Tool (GREAT) (McLean et al., 2010). Peak intersections and overlaps with differentially expressed genes were performed using Galaxy (Blankenberg et al., 2010) and in house shell scripts.
Genome_build: hg19
Supplementary_files_format_and_content: bedgraph files were created using Homer (Heinz et al., 2010) using -auto parameter converted into bigwig (bedGraphToBigWig) and visualized on UCSC (Kent et al., 2002) genome browser as custom tracks
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| Submission date |
Jan 27, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Lauren Goddard |
| Organization name |
University of California-Los Angeles
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| Department |
Molecular, Cell, and Developmental Biology
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| Lab |
Dr. Luisa Iruela-Arispe
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| Street address |
621 Charles E. Young Dr. S.
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| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (3) |
| GSE43786 |
Physiological Vascular Permeability Requires Induction of Endothelial NR4A1 by Progesterone Receptor [ChIP-Seq] |
| GSE43789 |
Physiological Vascular Permeability Requires Induction of Endothelial NR4A1 by Progesterone Receptor |
| GSE46503 |
Selective suppression of endothelial cytokine production by progesterone receptor |
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| Relations |
| SRA |
SRX220066 |
| BioSample |
SAMN01906481 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1071301_INPUT_PR_PR_infected+Prog.bw |
44.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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