|
| Status |
Public on Oct 01, 2015 |
| Title |
Tumors IL6 (MACS) |
| Sample type |
SRA |
| |
|
| Source name |
Pooled gastric tumor tissue from 3 individual IL6-treated gp130F/F mice, Stat3 ChIP
|
| Organism |
Mus musculus |
| Characteristics |
genotype/variation: gp130F/F
tissue: gastric tumors
age: 10-12 weeks
treatment: IL6
chip antibody: anti-Stat3
|
| Treatment protocol |
Mice (10-12 weeks, n=3 per treatment cohort) were subjected to a single intraperitoneal injection of PBS (200ul), IL6 or IL11 (5ug diluted in 200ul PBS) 60 min prior to sacrifice. Gastric tumor tissue was resected, snap-frozen on dry ice and stored at -80 degrees Celsius.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Following DNA-Protein cross-linking with formaldehyde and tissue homogenization, chromatin was extracted using "shearing buffer" provided in the SIGMA Imprint ChIP Kit in accordance with the manufacturer's instructions. Chromatin shearing was performed using the Diagenode Bioruptor water bath sonicator. Sheared chromatin was used for immunoprecipitations with a ChIP-validated Stat3 antibody (Santa Cruz SC-482X) and the SIGMA Imprint ChIP Kit according to the manufacturer's instructions.
Libraries were prepared according to Illumina's instructions accompanying the ChIP-Seq DNA Sample Preparation Kit (IP-102-1001). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow Exo fragment and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters, which have a single 'T' base overhang at the 3' end. Following adapter ligation, DNA was PCR amplified (15 cycles) with Illumina primers, and library fragments of 200-300 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer II following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
IL6
Chromatin
|
| Data processing |
Base-calling: Base-calling was performed using Illumina software. Reads that have poor sequence quality and/or are made of homopolymers were removed from the datasets.
Read alignment: After filtering of poor quality sequences, ChIP-Seq reads were aligned to the UCSC mm9 genome assembly using Bowtie software.
File conversion: Conversion of Bowtie output files to .bed and .wig files was performed using Perl.
Peak calling: Peak calling was performed using MACS software (Zheng et al., 2008) with a fold-enrichment ratio (mfold) of 12 fold and a standard threshold probability (p-value) of P≤10-5.
Retrieval of relevant peak and associated gene/microRNA information was performed using R programming language for statistical computing (Version 2.12.1).
Genome_build: MGSCv37
Supplementary_files_format_and_content: .bed and .xls files. The three processed data files "Control_Peak_Information_MACS_Output.xls", "IL6_Peak_Information_MACS_Output.xls" and "IL11_Peak_Information _MACS_Output.xls" are the original output files of the MACS software and contain the following information about significant Stat3-associated peaks: (I) exact peak location (chromosome number and the start and end nucleotide positions), (II) peak width/length (expressed in base pairs), (III) peak summit (the exact nucleotide position in each peak at which the highest number of tags were counted), (IV) reads/tags per peak (total number of sequence tags counted at each enriched region ), (V) P-value and (VI) fold enrichment of each peak.
|
| |
|
| Submission date |
Jun 25, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Stefan Thiem |
| E-mail(s) |
SteThiem@gmail.com
|
| Phone |
+61-3-9496-9775
|
| Organization name |
Olivia Newton-John Cancer Research Institute
|
| Department |
Cancer Research
|
| Lab |
Cancer & Inflammation Laboratory
|
| Street address |
145 Studley Road
|
| City |
Heidelberg - Melbourne |
| State/province |
Victoria |
| ZIP/Postal code |
3084 |
| Country |
Australia |
| |
|
| Platform ID |
GPL9250 |
| Series (2) |
| GSE48285 |
Genome-wide ChIP-Seq analysis of genomic Stat3 binding sites in murine gastric tumors following stimulation with IL6 or IL11 (MACS) |
| GSE48288 |
Genome-wide ChIP-Seq analysis of genomic Stat3 binding sites in murine gastric tumors following stimulation with IL6 or IL11 |
|
| Relations |
| Reanalyzed by |
GSM1174239 |
| BioSample |
SAMN02213665 |
| SRA |
SRX315095 |
