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| Status |
Public on Oct 20, 2017 |
| Title |
SC_internal SPEN_untreated |
| Sample type |
SRA |
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| Source name |
spermatocytes
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| Organism |
Mus musculus |
| Characteristics |
strain: FVB/N
Sex: male
age: adult (10-16 weeks)
tissue/cells: testes/spermatocytes
chip antibody: internal SPEN (Abcam, ab72266)
treatment: untreated control
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| Treatment protocol |
For heat shock, equal volumes of CO2 saturated, pre-heated media (to 53°C or 60°C) were added to the cells suspensions, which immediately raised their temperature from 32°C (physiological temperature) to 38°C or 43°C, respectively. Tubes were submerged in a water bath at the appropriate temperature for an additional 15-30 minutes with recovery at 32°C in a cell culture incubator.
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| Growth protocol |
Testicular cells for ChIP experiments were collected after collagenase type IA digestion of decapsulated testes (1mg/ml in CO2 saturated RPMI medium for 20 min at 32°C in a shaking water bath operated at 120 cycles/min). The dispersed seminiferous tubules were isolated by allowing them to sediment for 2-3 min under 5% CO2 in air and decanting the supernatant. The process was repeated 3 times with 10 ml RPMI to stop the reaction and to ensure removal of the dissociated interstitial cells and blood cells. Cell aggregates were dissociated gently by repeated pipetting for 3-5 min. Cells were suspended in CO2 saturated RPMI medium supplemented with 10% (v/v) fetal bovine serum, 0.004% (v/v) gentamycin (KRKA), and 6mM sodium lactate (Sigma) at 32°C (physiological temperature of testes). Population of cells enriched in spermatocytes (up to 80%) was obtained by unit gravity sedimentation of testicular cells obtained after enzymatic treatment (collagenase type IA, trypsin, and DNase I) in a Sta-Put chamber (SP-120, ProScience Inc, Ontario, Canada) containing a 1-3% (w/v) linear BSA gradient in PBS. Finally cells were suspended in RPMI medium as the mixture of testicular cells.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The ChIP assay was carried out according to the protocol of a ChIP kit from Upstate Biotechnology (Lake Placid, NY) using protein A-sepharose beads (Amersham). Standard crosslinking procedure was used. For each IP reaction 48-50 µg of chromatin sonicated to 100-500 bp fragments and 5 µg of anti-SPEN polyclonal antibodies, or IgG from rabbit serum (Sigma) as a negative control were used. Immunoprecipitated DNA was additionally digested by RNase A (in a final concentration 70 μg/ml, 30 minutes, 37°C) before Proteinase K treatment and organic solvents extraction. Glycogen (70 μg/ml) was added at the precipitation step. DNA was dissolved in H2O. In each experimental point five to six ChIP replicates were collected and combined in one sample before DNA sequencing.
Sequencing libraries were generated using KAPA library preparation kit (for Illumina) KK8201. Template amplification and cluster generation were performed using the TruSeq SBS Kit.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Reads were mapped to the mouse genome (mm10) with Bowtie2.
Aligned files were converted to bam and sorted using Samtools.
Peaks detection was carried with case-control strategy (detected peaks are given in bed files)
SPEN target sites were annotated to genomic regions using HOMER software (annotated binding sites are given in text file)
Genome_build: mm10
Supplementary_files_format_and_content: bed - detected peaks, can be open in text editors/Excel; txt - annotated binding sites, can be opened in text editors/Excel.
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| Submission date |
Jun 09, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Tomasz Stokowy |
| E-mail(s) |
tomasz.stokowy@uib.no
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| Organization name |
University of Bergen
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| Department |
IT Division
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| Lab |
Scientific Computing
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| Street address |
Nygardsgaten 5
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| City |
Bergen |
| ZIP/Postal code |
5020 |
| Country |
Norway |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE99856 |
Ability of SPEN to bind to chromatin in testicular cells |
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| Relations |
| BioSample |
SAMN07208494 |
| SRA |
SRX2899186 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2656052_SCK_1.txt.gz |
115.5 Kb |
(ftp)(http) |
TXT |
| GSM2656052_SCK_1_peaks.bed.gz |
26.0 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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