NextSeq 500 sequencing; ChIP-seq for TAZ (WWTR1) binding region in C2C12 myoblasts
ChIP-seq for TAZ (WWTR1) binding region in C2C12 myoblasts
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).
Experimental Factor: phenotype
mTAZ overexpression
Experimental Factor: immunoprecipitate
anti-FLAG