 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jan 26, 2021 |
| Title |
Round_spermatid_WT_IgG |
| Sample type |
SRA |
| |
|
| Source name |
Round, spermatid, WT, IgG
|
| Organism |
Mus musculus |
| Characteristics |
strain: 129 Sv/Ev
genotype: wild type
cell type: Round spermatid
chip antibody: anti-IgG (EMD Millipore, catalog #12-370)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The pachytene spermatocyte sample consisted of cellular suspensions obtained from testicular tubules isolated from eighteen day old male mice following our laboratory's standard protocols as previously published (PMID: 2441396). Briefly, testes were decapsulated and transferred to cold PBS, The tubules were manually sheared with scissors and by pipetting, and then passed through a 40 μm filter. Although this population also includes spermatogonia, leptotene/zygotene spermatocytes, and some Sertoli cells, these cells do not express BRDT. Round spermatids were purified as previously described (PMID: 2441396). Briefly, testes were dissected from seven 129 Sv/Ev adult mice and were decapsulated. The tubular mass was transferred to a 50 ml Erlenmeyer flask containing 25 ml of solution composed of 1.0 mg/ml collagenase (Sigma Cat# C0130) in RPMI buffer. The tubules were then incubated at 33oC in a shaking water bath for ~7 min until dispersed. They were then allowed to sediment to the bottom of the flask and the supernatant was removed by aspiration. Thetubules were washed twice with 25 ml of RPMI, then resuspended in 25 ml of RPMI containing 0.25 mg/ml trypsin (Sigma cat#T8003) and incubated at 33oC in a shaking water bath for 5 min. Cell aggregates and connective tissue were removed by filtration through 74-pm nylon mesh (BD Falcon). The single cell suspension of germ cells was separated using gravity sedimentation on a 2-4% BSA in DPBS gradient. Fractions containing round spermatids were pooled to yield samples containing ≥90% round spermatids, as assessed by flow cytometric analysis using propidium iodide (Sigma cat#P4170) staining and analysis on a BD FACSCalibur Cell Analyzer. All steps of cell separation, fractionation and pooling were performed at 4oC in order to maintain cell viability and maximize nucleic acid integrity.
To identify BRDT-containing complex binding sites, we prepared cross-linked chromatin samples for ChIP-Seq analysis. Approximately 107 cells from the two cellular preparations were fixed with 1% formaldehyde for 10 min at room temperature. The chromatin template was then fragmented to a size of 150 to 200 bp by sonication. Aliquots of the chromatin samples were subjected to chromatin immunoprecipitation using a C-terminal anti-BRDT antibody generated by our laboratory (PMID: 17728347) or anti-IgG as a control. This was followed by performing end repair and addition of “A” bases to the 3’ end of the DNA fragments, and ligation of adapters to the DNA fragments. The resulting fragments were then size-selected for 150-350bp regions before generating the library, which was enriched for the adapter-modified DNA fragments by PCR. The purified DNA samples were subjected to analysis on the Ilumina Cluster Station and Genome Analyzer.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Data processing |
Sequenced tags were mapped to the mouse genome (mm8) using Bowtie algorithm with the option ‘-m 1’. In order to reduce PCR amplification bias, at most one read was kept per genomic location. The resulting lists of uniquely mapped non-redundant reads were used for all downstream analyses. UCSC genome browser displayable BedGraph files for ChIP-Seq data were generated by counting tags in 200-bp windows across the genome and normalizing window tag counts by 10 million total sequenced reads to make different libraries directly comparable. BRDT binding sites from the ChIP-Seq data were identified using SICER software (PMID: 19505939) using IgG data as control and a False Discovery Rate (FDR) threshold of 10%.
Genome_build: mm8
Supplementary_files_format_and_content: BedGraph files contain normalized ChIP-Seq counts as described above. BED files contain locations of BRDT peaks.
|
| |
|
| Submission date |
May 03, 2017 |
| Last update date |
Jan 28, 2021 |
| Contact name |
Iouri Chepelev |
| E-mail(s) |
ichepelev@gmail.com
|
| Organization name |
US Department of Veterans Affairs Medical Center
|
| Street address |
3200 Vine St
|
| City |
Cincinnati |
| State/province |
OH |
| ZIP/Postal code |
45220 |
| Country |
USA |
| |
|
| Platform ID |
GPL9250 |
| Series (1) |
| GSE98489 |
Genome-wide analysis of the distribution of the testis-specific double bromodomain protein BRDT reveals distinct roles in pachytene spermatocyte and round spermatids |
|
| Relations |
| BioSample |
SAMN06885846 |
| SRA |
SRX2780263 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2597341_mm8_Round_spermatid_WT_IgG_GA1834.bedgraph.gz |
50.1 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
