|
| Status |
Public on Mar 06, 2012 |
| Title |
H2Bub_MB_replicate1 |
| Sample type |
SRA |
| |
|
| Source name |
Growing C1C12 myoblasts
|
| Organism |
Mus musculus |
| Characteristics |
cell type: C2C12
chromatin preparation method: MNAse digestion
chip antibody: H2Bub, Millipore: 05-1312, clone 56
antibody lot number: DAM1588299
|
| Treatment protocol |
Isolated chromatin was either sonicated to an average size of 250 bp or digested with MNAse to yield ~85% mono nucleososomal fragments.
|
| Growth protocol |
C1C12 myoblasts were grown in DMEM containing 10% FBS and harvested at ~40% confluency for MB chromatin. For myotube differentiation, cells were allowed to grow to complete confluence at which point differentiation was induced by changing the media to DMEM containing 2% horse serum. Induced cells were incubated for 4 days with one change of media (DMEM/2% horse serum) after two days and myotubes were harvested after 4 days of differentiation.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Libraries were generated from input chromatin or ChIP enriched chromatin according to Illumina's standard protocol but using reagents from other companies: DNA end-repair was performed using the End-It DNA repair kit, Epicentre (cat:ER81050). A-overhang generation was performed uising Klenow from NED (cat: M0212). Adapter ligation was performede using the LigaFast kit from Promega (cat:M8225). LM-PCR was performed using Phusion DNA pol master mix from NEB (cat:F-531S). Very stringent quality controls were applied to the libraries: 1. Enrichment of a given site had to be preserved in the libraries in a manner comparable to the original ChIP. 10-12 positive and negative sites were tested for library preparation. 2. The total yield of the LM-PCR had to be ~500 ng. 3. The size distribution had to be centred around 250 bp for libraries generated from MNAse chromatin and around 350 bp for sonicated with a variation no larger than +/- 75 bp. Libraries that did not pass these criteria were considered substandard and were not used for sequencing.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Data processing |
Sequence reads within each individual replicate were filtered to remove identical sequence tags since those were likely generated by the LM-PCR amplification process while identical sequences present after the merging of replicates were retained. The sequence reads were aligned against the mouse mm9 genome build using the Illumina Pipeline and only tags locating to a single unique position were retained.
Peaks were identified using Qeseq, an algorithm developed in collaboration with Dr Klugers bioinformatics group. Setting for all MNAse libraries: sigma=50 and cut-off=15. For sonicated libraries the settings used were: sigma=100 and cut-off=12.
|
| |
|
| Submission date |
Jan 09, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Brian Dynlacht |
| E-mail(s) |
brian.dynlacht@nyumc.org
|
| Organization name |
NYU Langone Medical Center
|
| Street address |
550 1st avenue
|
| City |
New York |
| ZIP/Postal code |
10016 |
| Country |
USA |
| |
|
| Platform ID |
GPL9250 |
| Series (1) |
| GSE34960 |
Dynamic loss of H2B ubiquitylation without corresponding changes in H3K4 tri-methylation during myogenic differentiation |
|
| Relations |
| SRA |
SRX115554 |
| BioSample |
SAMN00771673 |
