|
| Status |
Public on Oct 07, 2014 |
| Title |
HSF2_HS_38 |
| Sample type |
SRA |
| |
|
| Source name |
HSF2 ChIP DNA from spermatocytes heat shocked at 38°C
|
| Organism |
Mus musculus |
| Characteristics |
strain: FVB/N
gender: male
tissue: testis
cell type: spermatocytes
developmental stage: adult
age: 10-16 weeks
temperature: heatshock_38C
chip antibody: anti-HSF2 (cat. no AF5227, R&D Systems)
|
| Treatment protocol |
An equal volume of CO2 saturated, pre-heated to 53°C medium was added to the cell suspension, which immediately raised the temperature from 32°C to 38°C. The tubes were sealed with parafilm and submerged in a water bath at 38°C for an additional 5, 10, or 20 min. Samples were combined in one heat shocked sample.
|
| Growth protocol |
Testes (twenty males for one isolation) were decapsulated and cell suspension was obtained by enzymatic treatment (collagenase type IA, trypsin and DNase I). Population of cells enriched in spermatocytes (up to 80%) was obtained by unit gravity sedimentation 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 CO2 saturated RPMI medium supplemented with 10% (v/v) fetal bovine serum, 0.004% (v/v) gentamycin, and 6mM sodium lactate, at the temperature 32°C (physiological temperature of testes).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed by adding of formaldehyde to a final concentration of 1% and incubated at room temperature for 10 minutes. Next 2.5 M glycine was added (1/20 volume) for 5 minutes. Cells were washed with ice-cold PBS and lysed following the manufacturer’s protocol. For analyses of HSF1 and HSF2 binding, the ChIP assay was carried out according to protocol for ChIP kit of Upstate Biotechnology (Lake Placid, NY) and protein A-sepharose (Amersham) was used. For 30µg of chromatin, sonicated to 100-500 bp fragments, 3 µg of rabbit anti-HSF1 (cat. no SPA-901, Enzo) or 5 µg of goat anti-HSF2 (cat. no AF5227, R&D Systems) polyclonal antibodies were used. Samples proceeded with normal goat serum served as negative controls.
Sequencing libraries were generated using ChIP-Seq Sample Prep Kit (Illumina). Template amplification and cluster generation were performed using the cBot and TruSeq SR Cluster Kit v2 cBot-GA for cBot, and 80 nucleotides were sequenced with Illumina Genome Analyzer IIx using TruSeq SBS Kit v5 sequencing kits.
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| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
bibl11
HSF2 binding sites in spermatocytes heat shocked at 38°C
|
| 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)
HSF1 and HSF2 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; txt - annotated binding sites.
|
| |
|
| Submission date |
Apr 11, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Tomasz Stokowy |
| E-mail(s) |
tomasz.stokowy@uib.no
|
| Organization name |
University of Bergen
|
| Department |
IT Division
|
| Lab |
Scientific Computing
|
| Street address |
Nygardsgaten 5
|
| City |
Bergen |
| ZIP/Postal code |
5020 |
| Country |
Norway |
| |
|
| Platform ID |
GPL11002 |
| Series (1) |
| GSE56735 |
HSF1 and HSF2 interactions with chromatin during the heat shock response in mouse spermatocytes |
|
| Relations |
| BioSample |
SAMN02726025 |
| SRA |
SRX515500 |
