GSM1702885: MyoD (siMyolinc); Mus musculus; ChIP-Seq
After two days of differentiation, C2C12 cells were detached with trypsin-EDTA and fixed with 1% formaldehyde. Shearing of chromatin was carried out through Bioruptor Plus (Diagenode) using the following sonication steps: 20 cycles at the low intensity for 30 sec ON / 30 sec OFF; 40 cycles at the high intensity for 30 sec ON / 30 sec OFF; and 50 cycles at the high intensity for 30 sec ON / 30 sec OFF. The efficiency of shearing was assessed by running the sheared chromatin on an 2% agarose gel. Using this sheared chromation, ChIP was performing with EZ-Magna ChIP A/G Kit (Millipore, #17-10086) following manufacturer’s protocol. For each ChIP experiment, 10 μg of an antibody were used. For each library preparation, 100 ng of immunoprecipitated DNA were used as a starting template following manufacturer’s protocol. Briefly, immunoprecipitated DNA was end-repaired, adapter-ligated and amplified using Ion Plus Fragment Library Kit (Life Technologies, #4471252). The adapter-ligated DNA was size selected through E-Gel Electrophoresis System (Life Technologies) to obtain the optimal size for 200bp read sequencing. Then, size selected DNA was amplified for 18 cycles of PCR. After amplification, the fragments size of the library was assessed through High Sensitivity DNA Analysis Kit (Agilent, # 5067-4626). To calculate a dilution factor, Ion Library Quantitation Kit (Life Technologies, #4468802) was used. Using this dilution factor, template-positive Ion-Sphere particles were prepared using Ion PGM™ Template OT2 200 Kit (Life Technologies,  #4480974) via Ion OneTouch 2 System (Life Technologies). The quality of template-positive Ion-Sphere particles was assessed via Ion-Sphere™ Quality Control Assay (Life Technologies, #4468656). Particles were subsequently enriched according to manufacturer’s protocols. The enriched template-positive Ion-Sphere particles were sequenced using Ion 318™ Chip Kit v2 (Life Technologies, #4484355) and Ion PGM™ Hi-Q™ Sequencing Kit (Life Technologies, #A25592) via Ion PGM™ System (Life Technologies).
GEO Accession
GSM1702885