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| Status |
Public on Jun 17, 2016 |
| Title |
C2C12 differentiated Dam control 1 |
| Sample type |
SRA |
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| Source name |
myoblast
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| Organism |
Mus musculus |
| Characteristics |
treatment: differentiated
damid: Dam
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| Treatment protocol |
Myoblasts were induced to differentiate at 48h post-confluency by starvation using medium with 2% horse serum. Differentiation media was replaced every 48h up to 144h post-induction. To inhibit contraction 1uM tetrodotoxin was added at 96h post-induction,
Lentivirus encoding the control Dam, or LaminB1-Dam fusion, was used to transduce untreated myoblasts or differentiated myoblasts at 72h post-induction. Transduction was allowed for 24h. At 72h post-transduction genomic DNA was extracted.
Dam-methylated genomic DNA was enriched using the standard protocol as described by Vogel et al. (2007) Nature Protocols 2:1467-1478. It involves digesting gDNA with DpnI enzyme, which cuts at non-Adenine-methylated GATC motifs.
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| Growth protocol |
C2C12 Myoblasts (MBs) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal calf serum (FBS), 100 units/ml penicillin and 100 μg/ml streptomycin and passaged every 2 days in a 1:7 dilution by trypsinisation. Critically, cells were never permitted to reach more than 80% confluency, i.e. ~2.5 million cells on an 8.5 cm diameter tissue culture plate.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted using Qiagen DNA-Easy kit, following manufacturer's instructions.
Libraries were prepared by Beijing Genomics Institute (BGI) to Illumina's specifications
DamID
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
C2C12 differentiated Dam control
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| Data processing |
Basecalls and quality filtering performed by BGI according to their standard procedures
Clean reads were aligned to the mouse mm9 genome using the Burrows-Wheeler aligner bwa mem. Mitochondrial sequences (a common contaminant in these experiments) and unaligned reads were filtered out (output: ffSE_xx_mm9.bam files).
Reads were counted per DpnI fragment (genomic sequence flanked by GATC motifs), and log2(ratio LaminB1/dam) measurements were obtained for undifferentiated and differentiated samples. The log2 ratios were then normalised using quantile normalisation (Bioconductor Limma package) and converted to bigwig format for visualisation purposes.
To identify LADs, the peakfinder software SICER (v1.1) was employed with the following parameters: redundancy threshold 1, window size 500bp, gap size 0, fragment size 150bp, effective genome fraction 0.75, FDR 0.05
IP and PI regions were then identified by comparing the mean intensity differences between MT (differentiated C2C12) and MB (undifferentiated C2C12) log2(Lamin B1/Dam) values along a running 100 kb window. Regions with a mean MT/MB lamin B1 signal difference of 2 fold were then tested for significance against a randomized signal sample population using Fisher's exact test over 1,000 iterations. Regions with a P>0.01 were disregarded
Genome_build: mm9
Supplementary_files_format_and_content: sqq3_U.bw contains the DamID data for C2C12 undifferentiated as log2(LaminB1/dam control), per DpnI fragment, quantile normalised.
Supplementary_files_format_and_content: sqq3_D.bw contains the DamID data for C2C12 differentiated for 6 days as log2(LaminB1/dam control), per DpnI fragment, quantile normalised.
Supplementary_files_format_and_content: ffSE_32_mm9-W500-G0-FDR0.05-island.bed contains the coordinates of LaminB1 DamID peaks for C2C12 undifferentiated, obtained with SICER
Supplementary_files_format_and_content: ffSE_35_mm9-W500-G0-FDR0.05-island.bed contains the coordinates of LaminB1 DamID peaks for C2C12 differentiated (6 days), obtained with SICER
Supplementary_files_format_and_content: 150811_0.01_DR_U.bed contains the coordinates of differential regions (DR) that appear more associated with the nuclear envelope in the undifferentiated state (PI regions)
Supplementary_files_format_and_content: 150811_0.01_DR_D.bed contains the coordinates of differential regions (DR) that appear more associated with the nuclear envelope in the differentiated state (IP regions)
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| Submission date |
Apr 15, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Jose Ignacio de las Heras |
| E-mail(s) |
j.delasheras@ed.ac.uk
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| Organization name |
University of Edinburgh
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| Department |
Wellcome Trust Centre
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| Lab |
Swann 5.12
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| Street address |
Max Born Crescent
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| City |
Edinburgh |
| ZIP/Postal code |
EH9 3BF |
| Country |
United Kingdom |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE80328 |
LaminB1 DamID in undifferentiated mouse C2C12 myoblasts (ATCC, Lot 59501261) and differentiated C2C12 myotubes |
| GSE80330 |
Establishment of tissue-specific genome organisation by muscle-specific nuclear envelope transmembrane proteins (NETs) during mouse C2C12 myoblast differentiation |
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| Relations |
| BioSample |
SAMN04858621 |
| SRA |
SRX1707568 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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