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The cells were cross-linked with a final concentration of 1% formaldehyde followed by quenching with glycine. Cells were lysed with lysis buffer (0.2% SDS;10 mM Tris -HCl, pH 8.0; 10 mM EDTA, pH 8.0; proteinase inhibitor cocktail) and sonicated to fragments about 300 - 500 bp (Bioruptor, Diagenode). Dynabeads Protein A was washed twice with ChIP Buffer (10mM Tris-HCl pH7.5, 140mM NaCl, 1mM EDTA, 0.5mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, Cocktail proteinase inhibitor) and was incubated with antibody (ab8580 for H3K4me3, ab4729 for H3K27ac) at 4 °C for 2-3hours. The fragmented chromatin was transferred to the bead-antibody complex tubes and rotated at 4 °C overnight. The beads were washed once with low salt buffer (10mM Tris-HCl pH7.5, 250mM NaCl, 1mM EDTA, 0.5mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, Cocktail proteinase inhibitor) and twice with high salt buffer (10mM Tris-HCl pH7.5, 500mM NaCl, 1mM EDTA, 0.5mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, Cocktail proteinase inhibitor). |
ChIP-Seq |
GENOMIC |
PCR |
PAIRED
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