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Sample GSM1367511 Query DataSets for GSM1367511
Status Public on Oct 07, 2014
Title HSF2_C
Sample type SRA
 
Source name HSF2 ChIP DNA from untreated spermatocytes
Organism Mus musculus
Characteristics strain: FVB/N
gender: male
tissue: testis
cell type: spermatocytes
developmental stage: adult
age: 10-16 weeks
temperature: control
chip antibody: anti-HSF2 (cat. no AF5227, R&D Systems)
Treatment protocol untreated
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.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina Genome Analyzer IIx
 
Description bibl10
HSF2 binding sites in spermatocytes at physiological temperature
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 SAMN02726024
SRA SRX515499

Supplementary file Size Download File type/resource
GSM1367511_annotation_bibl10_13_131106.txt.gz 100.0 Kb (ftp)(http) TXT
GSM1367511_bibl10_13_comparative_peaks.bed.gz 18.1 Kb (ftp)(http) BED
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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