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Sample GSM4081759 Query DataSets for GSM4081759
Status Public on Oct 05, 2019
Title ChIP-seq_control
Sample type SRA
Source name untreated control
Organism Homo sapiens
Characteristics cell line: MCF7
tumor: adenocarcinoma
Sex: female
age: 69 years
tissue/cells: breast
chip antibody: HSF1 polyclonal antibody (Enzo, ADI-SPA-901)
Treatment protocol Cells were seeded on 10cm2 culture plates. Next day the medium was replaced into phenol-free DMEM/F12 medium supplemented with 5% dextran-activated charcoal-stripped FBS and 48 hours later cells were subjected to hyperthermia at 43°C for 15 minutes or 17β-estradiol was added to final concentration of 10nM for indicated time (1h and 2h).
Growth protocol The wild-type MCF7 cells were purchased from ATCC (HTB-22) and used as a primary model. Cells were grown in DMEM/F12 medium supplemented with 10% fetal bovine serum and antibiotic-antimycotic cocktail (penicillin, streptomycin, amphotericin) at 37 °C in a humidified 5% CO2 atmosphere.
Extracted molecule genomic DNA
Extraction protocol At the appointed experimental points culture medium was removed and fixation was performed using 1% formaldehyde in PBS for 12 min at RT with rotating. Formaldehyde fixation was quenched by glycine (125 mM final concentration) and then nuclei were isolated using buffers and protocol from iDeal ChIP-seq Kit for Transcription Factors (Diagenode). Chromatin of nuclei re-suspended in 200 µl was sheared using Bioruptor® PLUS combined with the Bioruptor® Water cooler & Single Cycle Valve (at HIGH power setting) with 20 cycles of 30 sec shearing followed by 30 sec of standby; chromatin fragments with approximate length 100-600 bp were obtained. Chromatin immunoprecipitation was carried out using the iDeal ChIP-seq Kit for Transcription Factors (Diagenode) with 30μg of chromatin and 3µl/sample of anti-HSF1 polyclonal antibody (ADI-SPA-901, Enzo) or without antibody (mock-IP), according to the manufacturer protocol.
Immunoprecipitated DNA fragments from six ChIP replicates was pooled. ChIP samples and input DNA were sequenced using the HiSeq 1500 system with TruSeq workflow (Illumina)
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 1500
Description untreated control
Data processing Quality control of reads was performed with FastQC software
Reads were mapped to the human genome (hg19) with Bowtie2.0.4
Peak detection was carried with the MACS program (detected peaks are given in bed files)
HSF1 target sites were annotated to genomic regions using HOMER software (annotated binding sites are given in text file)
Peak intersections and their genomic coordinates were found using Bedtools software
The significance of differences between control untreated cells and cells subjected to estrogen and hyperthermia was estimated using MACS software; the FDR=0.05 level was selected as the significance threshold.
Genome_build: hg19
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.
Submission date Sep 17, 2019
Last update date Oct 05, 2019
Contact name Tomasz Stokowy
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 GPL18460
Series (2)
GSE137558 Heat Shock Factor 1 (HSF1) acquires transcriptional competence under 17b-estradiol in ERa-positive breast cancer cells [ChIP-seq]
GSE137560 Heat Shock Factor 1 (HSF1) acquires transcriptional competence under 17b-estradiol in ERa-positive breast cancer cells
BioSample SAMN12771835
SRA SRX6858247

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data not provided for this record

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