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Sample GSM5452253 Query DataSets for GSM5452253
Status Public on Jul 12, 2022
Title siPAM-1 rep2 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 Oligonucleoties of siRNA against mouse PAM-1 and scrambled control were obtained from Ribobio Technologies (Guangzhou, China). siRNAs were transfected at 100nM into C2C12 using Lipofectamine 2000 (Life Technologies). 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-histone H3-K27 acetylation (Abcam, ab4729) 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)
GSE180068 seRNA PAM-1 interacts with Ddx5 to regulate skeletal muscle satellite cell activation and aging through trans regulation of Timp2 expression [siPAM-1 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 SAMN20207798
SRA SRX11439246

Supplementary file Size Download File type/resource
GSM5452253_siPAM-1_rep2_MB_H3K27ac.bw 209.3 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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