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| Status |
Public on May 13, 2013 |
| Title |
ChIP_control_GFPAb_C2C12_GFPonly |
| Sample type |
SRA |
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| Source name |
C2C12_GFP, GFP ChIP
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| Organism |
Mus musculus |
| Characteristics |
cell line: C2C12
treatment: doxycycline
technique: ChIP-sequencing
chip antibody: anti-GFP goat polyclonal
antibody vendor/provider: M. Vermeulen
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| Treatment protocol |
GFP or NICDGFP protein induction: the cells were induced for 8h with 2ug/ml doxycycline.
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| Growth protocol |
C2C12 cells were cultured in DMEM medium supplemented with 20% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen) at 37 °C in 5% CO2 atmosphere.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were trypsinized according to standard procedures. A cell suspension with cell density of 5 million/ml was made in cell medium. Cells were subsequently cross-linked for 30 min at RT in medium plus 1% formaldehyde and 1/14 volume of buffer A (0.1M NaCl, 1mM EDTA pH8, 0.5mM EGTA pH8, 50mM HEPES pH7.6), quenched with 0.125 M glycine and washed at 4°C with the following buffers: (i) PBS, (ii) buffer B: 0.25% Triton-X-100, 10mM EDTA pH8, 0.5mM EGTA pH8, 20mM HEPES pH7.6, (iii) buffer C: 0.15M NaCl, 1mM EDTA pH8, 0.5mM EGTA pH8, 50 mM HEPES pH7.6. During each washing step, cells were rotated for 10 min at 4°C and subsequently centrifuged for 7 min at 500g 4°C. Cells were then suspended in ChIP incubation buffer (0.15% SDS, 1% Triton-X-100, 0.15M NaCl, 1mM EDTA pH8, 0.5mM EGTA pH8, 20mM HEPES pH7.6, 1x protease inhibitor cocktail complete (Roche)) at 20 million/ml and sheared using a Bioruptor (Diagenode) with the following settings: high power, 30s on/off, 18 cycles. Sonicated chromatin was centrifuged for 5 min at 13000 rpm in eppendorf tubes, supernatant was snap-freezed in liquid nitrogen and stored at -80 °C or directly used in ChIPs. For one ChIP, 100 µl chromatin was incubated with 2 µg antibody, 20 µl 50% protein A/G bead (Santa Cruz), in 1x ChIP incubation buffer with 0.1% BSA in a total volume of 300 µl o/n at 4°C. Beads were then washed with the following buffers: twice with buffer1: 0.1% SDS, 0.1% NaDOC, 1% Triton-X-100, 0.15M NaCl, 1mM EDTA pH8, 0.5mM EGTA pH8, 20mM HEPES pH7.6, once with buffer2: 0.1% SDS, 0.1% NaDOC, 1% Triton-X-100, 0.5M NaCl, 1mM EDTA pH8, 0.5mM EGTA pH8, 20mM HEPES pH7.6, once with buffer3: 0.25M LiCl, 0.5% NaDOC, 0.5% NP-40, 1mM EDTA pH8, 0.5mM EGTA pH8, 20mM HEPES pH7.6, and twice with buffer4: 1mM EDTA pH8, 0.5mM EGTA pH8, 20mM HEPES pH7.6. During each washing step, beads were rotated for 5 min at 4°C and subsequently centrifuged for 2 min at 4000 rpm 4°C. Precipitated chromatin was then eluted from beads in 200 µl elution buffer (1% SDS, 0.1M NaHCO3) for 40 min at RT. Materials (precipitated chromatin or input chromatin in elution buffer) were decross-linked for 5h at 65 °C with addition of 200 mM NaCl. DNA was purified on QIAquick spin columns according to QIAGEN protocols. For sequencing, 6-8 ChIPs were pooled.
1 to 20 ng was used for library preparation using the ChIP-seq DNA sample Prep Kit (Illumina, cat# IP-102–1001) using the recommended protocol. After ligation of adapters, a 300 bp band is excised using the E-Gel system (Invitrogen), which correspond to a 234 bp fragment of genomic DNA. PCR enrichment of adapter-ligated DNA was done using 14 cycles of PCR.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
C2C12 cells expressing EGFP. C2C12 cells were transduced with a lentivirus Ubiquitin C-rTTA3 and a lenti tetO-EGFP. The lentiviral backbones are the same and derive from pTRIP-∆U3 lentivirus (for reference, see PMID 11319904). Only promoters and transgene were modified accordingly. To induce GFP expression, cells were induced for 8h with 2ug/ml doxycycline.
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| Data processing |
ChIP-seq: Cluster generation and sequencing-by-synthesis (36 bp) was performed using the Illumina Genome Analyzer IIx (GAIIx) platform according to standard protocols of the manufacturer (Illumina). The image files generated by the Genome Analyzer were processed to extract DNA sequence data. Sequences were aligned to the mouse (mm9) reference genome using the Illumina Analysis Pipeline, allowing one mismatch. Only the tags uniquely aligning to the genome were considered for further analysis. Duplicated reads were discarded to obtain a non-redundant set. Further analysis was performed using the 36 bp sequence reads. The output data were converted to Browser Extensible Data (BED) files for downstream analysis and Wiggle (WIG) files for viewing the data in the UCSC Genome Browser. All sequence analyses were conducted based on the Mus musculus mm9 genome assembly.
Genome_build: mm9
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| Submission date |
Apr 11, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Arjen Brinkman |
| E-mail(s) |
[email protected]
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| Organization name |
Radboud University, Nijmegen Center for Molecular Life Sciences
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| Department |
Molecular Biology
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| Lab |
Stunnenberg
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| Street address |
NCMLS #274, Geert Grooteplein Zuid 30
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| City |
Nijmegen |
| ZIP/Postal code |
6525 GA |
| Country |
Netherlands |
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| Platform ID |
GPL11002 |
| Series (1) |
| GSE37184 |
Dynamic binding of RBPJ is determined by Notch signalling status |
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| Relations |
| SRA |
SRX140345 |
| BioSample |
SAMN00854805 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM913300_ChIP_control_GFPAb_C2C12_GFPonly.reads.bed.gz |
172.6 Mb |
(ftp)(http) |
BED |
| GSM913300_ChIP_control_GFPAb_C2C12_GFPonly.wig.gz |
49.1 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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