NextSeq 500 sequencing; ChIP-seq for TAZ (WWTR1) binding region in C2C12 myoblasts
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
No description
Antigen
NA
Cell type
Cell type Class
Muscle
Cell type
C2C12
Primary Tissue
Skeletal Muscle
Tissue Diagnosis
NOS
Attributes by original data submitter
Sample
ENA first public
2018-08-15
ENA last update
2018-05-09
ENA-CHECKLIST
ERC000011
External Id
SAMEA4655421
INSDC center alias
Lab of stem cell and tissue regeneration, College of life sciences and biotechnology, Korea university
INSDC center name
Lab of stem cell and tissue regeneration, College of life sciences and biotechnology, Korea university
INSDC first public
2018-08-15T17:02:10Z
INSDC last update
2018-05-09T15:29:17Z
INSDC status
public
Submitter Id
E-MTAB-6764:C2C12-Bp
broker name
ArrayExpress
cell line
C2C12
cell type
myoblast
common name
house mouse
disease
normal
genotype
pBabe puro vector
organism part
muscle
phenotype
empty vector
sample name
E-MTAB-6764:C2C12-Bp
Sequenced DNA Library
library_name
C2C12-Bp_s
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Control and Flag-tagged TAZ-expressing C2C12 myoblasts were seeded and incubated for 24h in growing media (20% FBS-containing DMEM). Then cells were treated with 0.75% formaldehyde to cross-link proteins to DNA and quenched by 125 nM glycine. After rinse with PBS, cells were harvested and sonicated in RIPA buffer supplemented with protease inhibitor cocktail for chromatin fragmentation. After checking the fragment size by gel electrophoresis, chromatin samples were used for ChIP. Chromatin samples were immunoprecipitated with FLAG antibody-conjugated beads (Sigma, A2220). After washing, captured complexes were eluted and reverse cross-linking was performed to release DNA from protein-DNA complexes. Then, eluted DNA was purified by using DNA purification kit (Thermo scientific, K0832). Library preparation was performed with Illumina TruSeq ChIP sample preparation kit according to the manufacturer's instructions. Simply, After clean up of immunoprecipitated DNA using sample purification bead, the DNA was end-repaired at 30 °C for 30 min. A single 'A' nucleotide was added to the 3' ends of the blunt fragments using a-tailing mix reagent by incubation at 37 °C for 30 min, and then at 70 °C for 5 min. Indexing adapters were ligated to the ends of the DNA fragments using ligation mix 2 reagent at 30 °C for 10 min, followed by a gel size-selection for 250-350 bp of insert size. PCR was performed to enrich those DNA fragments that have adapter molecules on both ends. Thermocycler conditions were as follows: 98°C for 30 s, 18 cycles of 98°C for 10 s, 60°C for 30 s, and 72°C for 30 min, with a final extension at 72°C for 5 min. Finally, quality and band size of libraries were assessed using Agilent 2100 bioanalyzer (Agilent). Libraries were quantified by qPCR using CFX96 Real Time System (Biorad).