ChIP-Seq with no antibody in mouse M&M MEF cells transduced with GFP
cell type
mouse embryonic fibroblasts
genotype/variation
MyoD-/-; Myf5-/-
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Native ChIP for histone acetylation was performed by combining the nuclei isolation protocol from Gerber et al, 1997 and the MNase digestion and chromatin extraction method from Cao et al, 2010. Briefly: nuclei were harvested from 10 million cells by lysis in RSB+0.1% NP-40 for 10 min. Nuclei were rinsed with RSB and resuspended in 250 μl douncing buffer containing 15 U of MNase, and then incubated at 37°C for 18 min. The reaction was stopped by adding EDTA to 10 mM and moving the nuclei to ice. 1 ml of hypotonic lysis buffer with protease inhibitors was added to each sample. 50 μl of 0.25 M NaButyrate was added to each sample and the samples were rotated at 4°C for 1 hour. Insoluble material was removed by centrifugation (3800 xg for 5 min, 4°C). 100 μl of the supernatant (soluble chromatin) was removed and reserved for input DNA. No antibody was added to 1 ml of soluble chromatin and rotated overnight at 4°C. Nucleosome-antibody complexes were isolated with protein A:G agarose beads in the same manner as the xChIPs. ChIP samples were validated by qPCR and prepared for sequencing per the Nugen Ovation Ultralow library system with direct read barcodes.