title: GSM3454527: MYOD1 ChIPseq; Mus musculus; ChIP-Seq
library_construction_protocol: Approximately 800 mg of adult skeletal gastrocnemius muscle was homogenized in cell lysis buffer (10mM HEPES, pH7.5, 10mM MgCl2, 60mM KCl, 300mM sucrose, 0.1mM EDTA, pH8.0, 0.1% Triton X-100, 1mM DTT, protease inhibitors) containing 1% formaldehyde. Cross-linking was stopped after 30 minutes by adding glycine to a final concentration of 0.125M. Nuclei and muscle fibers were separated by centrifugation at 25g for 5 minutes. The supernatant was collected, and nuclei were spun down by centrifugation at 800 g for 5 minutes and re-suspended in 350 µl of RIPA buffer (20mM Tris, pH8.0, 2mM EDTA, pH8.0, 0.1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 140mM NaCl). The nuclei preparation was sonicated to shear the DNA to 150-600 bp and then centrifuged at 14,000 g for 15 minutes. The supernatant was collected and pre-cleared with protein A/G slurry at 4ºC for 1 hr.  Protein A/G beads were removed by centrifugation and supernatant was split into two equal parts, which will be mixed with either MYOD1 Ab or pre-immune serum and incubated overnight at 4ºC. The immune-complex was incubated with protein A/G beads slurry for 4 hours and the beads will be washed with RIPA buffer 5 times and TE buffer twice. DNA was then eluted twice with 250 µl of elution buffer (1% SDS and 0.1M NaHCO3) for 20 minutes and supernatant was collected after 5 minutes centrifugation at high speed. Twenty-five microliters of 4M NaCl was mixed with the 500µl of elute and incubated at 65ºC overnight. Then 10µl of 0.5M EDTA, 20µl of 1M Tris pH 6.5 and 2µl 10mg/ml proteinase K was added to each sample and incubated at 55ºC for 1.5 hr. DNA was recovered by using PCR purification kit (Qiagen). ChIP DNA samples from the MYOD1 antibody were prepared for Solexa sequencing according to the Illumina protocol using methods described in Cao et al., (Dev. Cell, 2010) with DNA fragments ranging in size from 150 to 600 bp.
attributes.tag: GEO Accession
attributes.value: GSM3454527