|
| Status |
Public on Jul 12, 2022 |
| Title |
PAM-1 KO rep1 H3K27ac ChIP-seq |
| Sample type |
SRA |
| |
|
| Source name |
C2C12 myoblast
|
| Organism |
Mus musculus |
| Characteristics |
cell type: C2C12 myoblast
strain: none
antibody: H3K27ac (ab4729, Abcam)
|
| Treatment protocol |
To delete PAM-1 exon1, target-specific guide RNAs (gRNAs) were designed using CRISPR design tool (http://crispr.mit.edu), followed by cloning into BbsI digested px330 plasmid (Addgene, 42230). To perform genomic deletion, a pair of gRNAs containing plasmids were co-transfected into C2C12 cells with screening plasmid pSIREN-RetroQ (Clontech) using Lipotectamine 2000. Cells were selected with 2.5ug of puromycin for 3 days at 48 hours post-trasnsfection. Cells were diluted to 1 cell per well in 96 well plate. Individual colonies were PCR validated.
|
| Growth protocol |
Mouse C2C12 myoblast cell (CRL-1772) was obtained from American Type Culture Collection (ATCC) and cultured in DMEM medium with 10% fetal bovine serum, 1% penicillin/ streptomycin at 37°C in 5% CO2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
C2C12 cells were crosslinked with 1% formaldehyde at room temperature for 10 minutes, followed by quenching with 0.125M glycine for 10 minutes. Chromatin was fragmented using S220 sonicator (Covaris), followed by incubation with 5ug of antibodies and 50uL Dynabeads Protein G magnetic beads (Life Technologies) at 4°C on rotator for overnight. Anti Ddx5 (ab21696, Abcam) was used in ChIP assay. Beads were washed with 1mL RIPA buffer for 5 times, followed by decrosslink at 65°C for 16 hours and DNA extraction with phenol/chloroform. Immunoprecipitated DNA was resuspended in 50uL of water.
NEBNext Ultra II DNA Library Preparation kit for Illumina (NEB)
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
NextSeq 550 |
| |
|
| Data processing |
The raw data were first preprocessed by initial quality assessment, adaptors trimming, and low-quality filtering
The remaining reads were aligned to the mouse reference genome (mm9) by Bowtie2
Peak regions were called by software MACS2
Genome_build: mm9
Supplementary_files_format_and_content: bigwig
|
| |
|
| Submission date |
Jul 14, 2021 |
| Last update date |
Jul 14, 2022 |
| Contact name |
Yile Huang |
| E-mail(s) |
huangyilekuaile@gmail.com
|
| Phone |
56289467
|
| Organization name |
The Chinese University of HongKong
|
| Department |
Chemical Pathology
|
| Street address |
Room 503, Li Ka Shing Health and Science Buiding, Prince of Wales Hospital, Sha Tin
|
| City |
Sha Tin |
| State/province |
Hong Kong |
| ZIP/Postal code |
999077 |
| Country |
China |
| |
|
| Platform ID |
GPL21626 |
| Series (2) |
| GSE180069 |
seRNA PAM-1 interacts with Ddx5 to regulate skeletal muscle satellite cell activation and aging through trans regulation of Timp2 expression [PAM-1 KO ChIP-seq] |
| GSE180073 |
seRNA PAM-1 interacts with Ddx5 to regulate skeletal muscle satellite cell activation and aging through trans regulation of Timp2 expression |
|
| Relations |
| BioSample |
SAMN20207769 |
| SRA |
SRX11439228 |
