|
| Status |
Public on Mar 01, 2016 |
| Title |
H4Ac_GFP |
| Sample type |
SRA |
| |
|
| Source name |
ChIP-Seq with H4Ac antibody 39925 in mouse M&M MEF cells transduced with GFP
|
| Organism |
Mus musculus |
| Characteristics |
cell type: mouse embryonic fibroblasts
genotype/variation: MyoD-/-; Myf5-/-
|
| Treatment protocol |
M&M MEF cells were infected in DMEM + 10% FBS media containing polybrene at a final concentration of 8 µg /ml. After 12 hours, media was replaced with fresh DMEM +10% FBS, and after 24 hours (from infection) the cells were transitioned to Differentiation media (DMEM containing insulin and transferin). Cells were harvested after 20 hours in Differentiation media. Negative controls for MyoD and Myf5 ChIPs were cells infected with lenti-GFP.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Native ChIP for histone acetylation was performed by combining the nuclei isolation protocol from Gerber et al, 1997 and the MNase digestion and chromatin extraction method from Cao et al, 2010. Briefly: nuclei were harvested from 10 million cells by lysis in RSB+0.1% NP-40 for 10 min. Nuclei were rinsed with RSB and resuspended in 250 μl douncing buffer containing 15 U of MNase, and then incubated at 37°C for 18 min. The reaction was stopped by adding EDTA to 10 mM and moving the nuclei to ice. 1 ml of hypotonic lysis buffer with protease inhibitors was added to each sample. 50 μl of 0.25 M NaButyrate was added to each sample and the samples were rotated at 4°C for 1 hour. Insoluble material was removed by centrifugation (3800 xg for 5 min, 4°C). 100 μl of the supernatant (soluble chromatin) was removed and reserved for input DNA.
20 μl of antibody was added to 1 ml of soluble chromatin and rotated overnight at 4°C. Nucleosome-antibody complexes were isolated with protein A:G agarose beads in the same manner as the xChIPs. ChIP samples were validated by qPCR and prepared for sequencing per the Nugen Ovation Ultralow library system with direct read barcodes.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Basecalls performed using CASAVA version 1.8.2
Reads were aligned to human genome hg19 using BWA (0.7.5)
Peaks were called using in-house peak calling algorithm
Genome_build: mm9
Supplementary_files_format_and_content: ChIP-Seq peaks in csv format
|
| |
|
| Submission date |
Nov 24, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Stephen Tapscott |
| E-mail(s) |
stapscot@fredhutch.org
|
| Organization name |
Fred Hutch Cancer Research Center
|
| Department |
Human Biology
|
| Lab |
Tapscott
|
| Street address |
1100 Fairview N. Ave
|
| City |
Seattle |
| State/province |
WASHINGTON |
| ZIP/Postal code |
98103 |
| Country |
USA |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE75370 |
Distinct activities of Myf5 and MyoD indicate sequential roles in skeletal muscle lineage specification and differentiation (ChIP-Seq) |
| GSE75632 |
Distinct activities of Myf5 and MyoD indicate sequential roles in skeletal muscle lineage specification and differentiation |
|
| Relations |
| BioSample |
SAMN04293440 |
| SRA |
SRX1451509 |
