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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Apr 06, 2018 |
| Title |
Pol2 |
| Sample type |
SRA |
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| Source name |
C2C12 cells, Pol2
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| Organism |
Mus musculus |
| Characteristics |
cell line: myoblast cell line C2C12
time: Day 2 of differentiation
chip antibody: RNA polymerase II CTD repeat YSPTSPS (phospho S2) (Abcam, catalog# ab5095; lot# GR193165-1)
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| Treatment protocol |
Transient siRNA transfection (10nM final concentration) was carried out using RNAiMax (Life Technologies) according to the manufacturer’s protocol. To induce the differentiation process (initiated 24 hours after the transfection), C2C12 cells were cultured in the differentiation medium containing 2% horse serum (Sigma-Aldrich, #H1270) in DMEM with low glucose, pyruvate (Life Technologies, #31885-023) supplemented with antibiotics (penicillin and streptomycin, Sigma-Aldrich, #P4333).
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| Growth protocol |
Murine myoblast cell line C2C12 cells were cultured in the growth medium containing 20% FBS (Life Technologies, #10270106) in DMEM with high glucose, pyruvate (Life Technologies, # 41966-029) supplemented with antibiotics (penicillin and streptomycin, Sigma-Aldrich, #P4333) at 37°C in a humidified atmosphere containing 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
After two days of differentiation, C2C12 cells were detached with trypsin-EDTA and fixed with 1% formaldehyde. Shearing of chromatin was carried out through Bioruptor Plus (Diagenode) using the following sonication steps: 20 cycles at the low intensity for 30 sec ON / 30 sec OFF; 40 cycles at the high intensity for 30 sec ON / 30 sec OFF; and 50 cycles at the high intensity for 30 sec ON / 30 sec OFF. The efficiency of shearing was assessed by running the sheared chromatin on an 2% agarose gel. Using this sheared chromation, ChIP was performing with EZ-Magna ChIP A/G Kit (Millipore, #17-10086) following manufacturer’s protocol. For each ChIP experiment, 10 μg of an antibody were used.
For each library preparation, 100 ng of immunoprecipitated DNA were used as a starting template following manufacturer’s protocol. Briefly, immunoprecipitated DNA was end-repaired, adapter-ligated and amplified using Ion Plus Fragment Library Kit (Life Technologies, #4471252). The adapter-ligated DNA was size selected through E-Gel Electrophoresis System (Life Technologies) to obtain the optimal size for 200bp read sequencing. Then, size selected DNA was amplified for 18 cycles of PCR. After amplification, the fragments size of the library was assessed through High Sensitivity DNA Analysis Kit (Agilent, # 5067-4626). To calculate a dilution factor, Ion Library Quantitation Kit (Life Technologies, #4468802) was used. Using this dilution factor, template-positive Ion-Sphere particles were prepared using Ion PGM™ Template OT2 200 Kit (Life Technologies, #4480974) via Ion OneTouch 2 System (Life Technologies). The quality of template-positive Ion-Sphere particles was assessed via Ion-Sphere™ Quality Control Assay (Life Technologies, #4468656). Particles were subsequently enriched according to manufacturer’s protocols. The enriched template-positive Ion-Sphere particles were sequenced using Ion 318™ Chip Kit v2 (Life Technologies, #4484355) and Ion PGM™ Hi-Q™ Sequencing Kit (Life Technologies, #A25592) via Ion PGM™ System (Life Technologies).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Ion Torrent PGM |
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| Data processing |
The sequencing reads were analyzed and mapped to the mouse genome assembly mm10 via Ion PGM Torrent Server (Life Technologies).
The BAM files were used as inputs to HOMER (Heinz et al., 2010) for peak calling and further analysis for motif discoveries.
Genome_build: mm10
Supplementary_files_format_and_content: Text files generated by HOMER.
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| Submission date |
Jun 03, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Shizuka Uchida |
| E-mail(s) |
[email protected], [email protected]
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| Organization name |
Aalborg University
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| Department |
Department of Clinical Medicine
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| Lab |
Center for RNA Medicine
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| Street address |
Frederikskaj 10B, 2. (building C)
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| City |
Copenhagen SV |
| ZIP/Postal code |
DK-2450 |
| Country |
Denmark |
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| Platform ID |
GPL16331 |
| Series (2) |
| GSE69527 |
An Evolutionarily-Conserved Long Noncoding RNA Myolinc Regulates muscle differentiation [ChIP-seq] |
| GSE69530 |
A novel long non-coding RNA Myolinc regulates myogenesis through TDP-43 and Filip1 |
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| Relations |
| BioSample |
SAMN03759856 |
| SRA |
SRX1047652 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1702876_Pol2_vs_IgG-peaks-annot.txt.gz |
841.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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