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| Status |
Public on Feb 17, 2017 |
| Title |
muscle_Pol II_WT |
| Sample type |
SRA |
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| Source name |
ChIP DNA
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| Organism |
Mus musculus |
| Characteristics |
strain background: C57/B6
genotype/variation: wild type
tissue: Hind-limb muscles
chip antibody: Pol II
chip antibody vendor: Santa Cruz
chip antibody cat. #: sc-9001 X
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Hind-limb muscles from wild-type and mutant (HSA::Cre-ERT2::Tead4lox/lox , Conditional muscle-specific Tead4 knockout) mice were minced and homogenised, fixed in 1% formaldehyde for 10 min at room temperature. Cross-linking was stopped by the adding Glycine to final concentration 0.125 M. The tissue lysate was further homogenized in hypotonic buffer in order to obtain nuclei, the nuclear pellet was washed once in hypotonic buffer and then resuspended in sonication buffer. Nuclear lysates were sonicated using Covaris E220 to obtain DNA fragments <500 bp. Chromatin was pretreated with blocked protein G sepharose beads. Subsequently, samples were incubated overnight at 4°C with antibodies to AcH3K27 (Active Motif) or RNA PoI II antibody (Santa cruz). Blocked beads were then added and the mixture was incubated for 2 h at 4°C. Prot G beads were washed, protein-DNA complexes were eluted from the beads and decrosslinked, and DNA was recovered by phenol chloroform extraction and ethanol precipitation after treatment with ribonuclease A (Abcam) and proteinase K.
ChIP-seq libraries were prepared using NEXTflex ChIP-Seq Kit (#5143-02, Bioo Scientific) following the manufacturer's protocol (V12.10) with some modifications. Briefly, 10 ng of ChIP enriched DNA or INPUT DNA were end repaired using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK, then size selected and cleaned-up using Agencourt AMPure XP beads (#A63881, Beckman). 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 barcoded DNA adapters (NEXTflex ChIP-Seq Barcodes - 6, #514120, Bioo Scientific) using T4 DNA Ligase. The ligated products were enriched by PCR (2 min at 98°C; [30 sec at 98°C, 30 sec at 65°C, 60 sec at 72°C] x 14 cycles; 4 min at 72°C) and cleaned-up using Agencourt AMPure XP beads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
SPJH71
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| Data processing |
Image analysis and base calling were performed using RTA 2.7.3 and bcl2fastq 2.17.1.14.
Sequence reads were mapped to reference genome mm9 using Bowtie 1.0.0 with the following parameters -m 1 --strata --best -y -S -l 40 -p 2.
Genome_build: mm9
Supplementary_files_format_and_content: Wig files were generated using with an in-house script (Variable step, span=25, reads were elongated to 200b).
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| Submission date |
Jun 29, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Shilpy Joshi |
| E-mail(s) |
shilpy.joshi@gmail.com
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| Organization name |
IGBMC
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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 |
GPL21103 |
| Series (2) |
| GSE82190 |
Specific and redundant roles of TEAD transcription factors in C2C12 cell and primary myoblast differentiation (ChIP-Seq) |
| GSE82193 |
Specific and redundant roles of TEAD transcription factors in C2C12 cell and primary myoblast differentiation |
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| Relations |
| BioSample |
SAMN05324085 |
| SRA |
SRX1884839 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2219814_wigs_for_SPJH71.wig.gz |
147.7 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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