GSM5799283: CCRF-CEM cells - H3K4me3 - 3D - rep3; Homo sapiens; ChIP-Seq
Cells were washed, strained and fixed with 1% formaldehyde for 4 min and quenched with 0.125 M glycine.  Lysates were sonicated with a QSonica to shear the DNA. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. For each ChIP reaction, 30 ug of chromatin was precleared with protein A agarose beads (Invitrogen). ChIP reaction was set up using precleared chromatin and antibody and incubated overnight at 4º C. Protein A agarose beads were added and incubation at 4 C was continued for another 3 hr. Immune complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65 C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. (1)DNA-endrepair,3'-dAoverhangandligationofmethylatedsequencingadaptor. (2) PCR amplification and size selection (usually 100-300bp, including adaptor sequence). (3)Qualified library for next generation sequencing.