Input for ChIP087.H3K4me3_siNS and ChIP088.H3_siNS
cell line
C2C12
cell condition
myoblasts
library protocol
ChIP-seq
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Preparation of solubilized chromatin fraction and immunoprecipitation: Cell pellets were resuspended in buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 1 mM dithiothreitol (DTT), and protease inhibitors (aprotinin, leupeptin, pepstatin A, and phenylmethyl sulphonyl fluoride)), supplemented with Triton X-100 (0.1%), and incubated on ice for 5 minutes. The nuclear pellet was separated from the cytoplasmic fraction, washed once with buffer A, and collected by centrifugation. Nuclei were lysed with buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM dithiothreitol (DTT), and protease inhibitors (aprotinin, leupeptin, pepstatin A, and phenylmethyl sulphonyl fluoride)). The solubilized chromatin fraction was separated from nucleoplasm by centrifugation at 2250 g for 4 minutes at 4°C, washed once with buffer B, and collected by centrifugation. For immunoprecipitations, the chromatin pellet was resuspended with binding buffer (20 mM Hepes pH7.9, 100 mM potassium chloride, 0.2 mM EDTA, 20% glycerol, 0.5 mM dithiothreitol (DTT), and 0.5mM AEBSF). 1 mg of protein was immunoprecipated, and antibody-protein A sepharose beads were washed three times with buffer (50 mM Hepes pH7.9, 250 mM sodium chloride, 5 mM EDTA, 0.50% NP-40, and 10% glycerol) prior to SDS–PAGE and immuno-blotting.