GSM2837688: C2C12 GM1 IP; Mus musculus; ChIP-Seq
Chromatin from 30 X 106 C2C12 cells was cross-linked with 1% formaldehyde (Sigma-Aldrich) for 12 minutes at room temperature.  Glycine was then added to a final concentration of 0.125 M for 5 minutes. After washing, samples were centrifuged and suspended in cell lysis buffer (10mM Tris pH 8.0, 10mM NaCl, 0.2% NP40), incubated on ice for 30 minutes and centrifuged at 4000 rpm for 5 min. Nuclei were then suspended in nuclei lysis buffer (50 mM Tris-HCl pH 8.1, 1% SDS, 10 mM EDTA) and incubated on ice for 30 minutes. To obtain chromatin fragments of around 200–300 bp, sonication was performed using a Bioruptor UCD-200 sonicator (Diagenode). Chromatin extracts were incubated overnight on a rotating platform at 4 °C with 7.5 μg anti-HDAC4 (Santa Cruz, sc-11418) for immunoprecipitation. Rabbit IgG was used as negative control. Immunoprecipitated chromatin was conjugated with G-protein magnetic Beads (Invitrogen). After washing, bound DNA fragments were eluted by adding elution buffer (1% SDS, 1mM EDTA, 10mM Tris). Purified DNA and input DNA were sequenced at the NextGen Sequencing facility (IGA Technologies Service, Udine). 'Ovation Ultralow Library System v2' kit (NuGEN, San Carlos, CA) has been used for library preparation following the manufacturer's instructions. Both input and immunoprecipitated samples as well as final libraries were quantified by using the Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and quality tested by Agilent 2100 Bioanalyzer High Sensitivity DNA assay (Agilent technologies, Santa Clara, CA) and Real Time Stratagen Mx3000P  (Agilent technologies, Santa Clara, CA).
GEO Accession
GSM2837688