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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 11, 2014 |
| Title |
HA-Ctrl_ChIP-Seq |
| Sample type |
SRA |
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| Source name |
pancreatic beta cell line
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| Organism |
Mus musculus |
| Characteristics |
cell type: Min6b1 cells
chip antibody: HA 12CA5
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| Growth protocol |
Min6b1 cells were grown in DMEM with 25mM glucose supplemented With 15% Foetal calf serum, penicillin/streptomycin and 71μM β-mercaptoethanol, in a 5% CO2 incubator at 37°C. Min6b1 were transfected with 24μg of pCMV-Tag2A-3HA-Rfx6 plasmid using Lipofectamine 2000 (Invitrogen) and harvested two days later for anti-HA (12CA5) ChIP.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with antibody.
ChIP-seq libraries were prepared using Truseq ChIP-Seq Sample Prep Kit (Illumina, IP-202-1012) following the manufacturer's protocol with some modifications. Briefly, 10 ng of ChIP enriched DNA or control DNA were end repaired using T4 DNA polymerase, Klenow DNA polymerase and T4 Poly Nucleotide Kinase . A single ‘A’ nucleotide was added to the 3’ ends of the blunt DNA fragments with a Klenow fragment (3' to 5'exo minus). The ends of the DNA fragments were ligated to double stranded adapters using T4 DNA ligase. The ligated products were enriched by PCR (30 sec at 98°C [10 sec at 98°C, 30 sec at 65°C, 30 sec at 72°C] x 14 cycles; 5 min at 72°C), then size selected and purified using Agencourt AMPure XP beads (#A63881, Beckman). Prior to analyses DNA libraries were checked for quality and quantified using a 2100 Bioanalyzer (Agilent). The libraries were loaded in the flowcell at 7pM concentration and clusters were generated using the Cbot (Illumina, SY-301-2002 ) and sequenced on the Illumina Genome Hiseq2500 as single-end 50 base reads following Illumina’s instructions
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Base calling : Image analysis and base calling were performed using RTA 1.17.20 and CASAVA 1.8.2
Alignment : Reads were mapped to mm9 assembly of mouse genome using Bowtie aligner release<=1.0.0, with principle parameters: -m 1 --strata --best
Peak calling : Peaks were called using the Model-based Analysis of ChIP-Seq (MACS 1.4.2) algorithm. Small dataset(HA-Ctrl_ChIP-Seq) was scaled towards larger dataset(HA-Rfx6_ChIP-Seq).
Wig files: For all samples, aligned sequences were extended to 200bp downstream (with respect to read strand) and allocated into 25bp bins.
Genome_build: mm9
Supplementary_files_format_and_content: MACS 1.4.2 peak calling file with the following collumns: chr, start,end,length,summit,tags,-10*LOG10(pvalue),fold_enrichment,FDR(%),peak name.
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| Submission date |
Oct 30, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Tao YE |
| Organization name |
IGBMC (CNRS/INSERM/UDS)
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| Street address |
1 rue Laurent Fries
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| City |
Illkirch |
| ZIP/Postal code |
67404 |
| Country |
France |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE62844 |
Rfx6 target genes in the Min6b1 cell line, a pancreatic beta cell model |
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| Relations |
| BioSample |
SAMN03153298 |
| SRA |
SRX747492 |
| Named Annotation |
GSM1534426_PSR-07_HA-Ctrl.wig.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1534426_PSR-07_HA-Ctrl.wig.gz |
66.2 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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