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| Status |
Public on Mar 06, 2015 |
| Title |
Brd4-1_round_spermatid |
| Sample type |
SRA |
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| Source name |
Round spermatid
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| Organism |
Mus musculus |
| Characteristics |
antibody: Bethyl A301-985A50
cell type: round spermatid
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| Growth protocol |
Male 129S6/SvEvTac mice (Taconics, Germantown,NY) as well as Hrb-/- gene-disrupted mice (Kang-Decker 2001) were maintained according to the guidelines of the University of Pennsylvania Institutional Animal Care and Use Committee.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Spermatogenic cell fractionation was performed by sedimentation of cells prepared from adult mouse testes through a BSA gradient as previously described (Bryant et al. 2013). Each fractionation experiment used approximately 22 testes. Fractions were analyzed for purity based on cell and nuclear morphology (via DAPI staining) and pooled. Mature spermatozoa were obtained from epididymides of adult mice, and contaminating cell types were eliminated by incubating in somatic cell lysis buffer (0.1% SDS, 0.5% Triton X-100 in DEPC H2O) on ice for 20 minutes.
Cells were cross-linked in 1% formaldehyde in PBS for 10 minutes at room temperature. The reaction was quenched with 125mM glycine in PBS for five minutes at room temperature. After cell lysis, lysates were sonicated for 20 minutes with a Covaris S220 sonicator (5% duty cycle, 140 watts peak incident power, 200 cycles per burst). For each IP, 500μg of protein (measured with BCA assay) from the cell lysate, 30uL protein G Dynabeads, and 5μg-10ug of antibody or IgG (Pierce 31235) were used. ChIP libraries for sequencing were prepared using 5ng DNA and the NEBNext Ultra DNA library prep kit for Illumina. Size selection was performed using AMPure XP beads (Beckman Coulter, Inc. #A63881). Libraries were sequenced using a NextSeq 500 machine (Illumina) as per manufacturer's protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Brd4 in round spermatids
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| Data processing |
Bases called with CASAVA, most recent version, and demultiplexed with bcl2fastq 2.0
Alignment: bowtie v0.12.7 with parameters --best and -m 1 for all samples
PCR duplicate filtering: for any given genomic position (chromosome,start,stop,strand), multiple tags with identical parameters were collapsed to a single representative
Peak-calling: SICER was used to call peaks in all chIP-seq data using input as a control for local sonication bias. Window, fragment, and gap size parameters were fixed at 200bp; and the FDR was controlled at 0.1%.
Track creation: tracks were generated by using the BEDTools utility genomeCoverageBed (-bg) to convert BED files to coverage maps (bedGraphs). These were then normalized to a scalar coefficient to correct for sequencing bias in any sample. The normalized input map was then subtracted from each chIP-seq map. Resulting bedGraphs were used to generate bigWigs via the bedGraphToBigWig utility in the UCSC Genome Browser tool suite.
Genome_build: NCBI v37
Supplementary_files_format_and_content: Peak files (BED format) are SICER output files. BigWigs are provided for visual inspection of the data.
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| Submission date |
Oct 03, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Gregory Donahue |
| Organization name |
The University of Pennsylvania
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| Department |
Cell & Developmental Biology
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| Lab |
Zaret Lab
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| Street address |
3400 Civic Center Blvd, Bldg 421
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE56526 |
BRD4 dynamics reveal novel acrosome-dependent chromatin reorganization during post-meiotic mammalian sperm development |
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| Relations |
| BioSample |
SAMN03093442 |
| SRA |
SRX719833 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1519002_Brd4-Input.mm9.bw |
618.8 Mb |
(ftp)(http) |
BW |
| GSM1519002_Brd4.Be.unique-W200-G200-FDR0.001-island.bed.gz |
143.8 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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