Homo sapiens Epigenomics

The DHS assays, briefly, primary SAEC, Lu-iPSC and 4 SCLC cell lines were grown to 75% confluency, harvested and washed in PBS. Nuclei were isolated after a brief treatment with 1% NP-40. Extracted chromatin was digested with different concentrations of DNase I (10U/ml to 100 U/ml) for 3 minutes at 37C. The reaction was stopped by adding equal volume of Stop buffer (containing spermine/spermidine/RNase A). All digestion tubes were incubated at 55C for 6 hours and Proteinase K (final concentration of 50 ug/ml) was added for further overnight incubation. The quality of digestion was analyzed by qPCR to determine which samples meet 75-85% digestion optimal for sequencing (19). DNA was purified by phenol-chloroform extraction and fragmented DNA was separated using a sucrose gradient. Fragments between 100bp and 500bp were isolated, DNA was precipitated, the samples were pooled into two biological replicates and sent for sequencing to Frederick National Laboratory for Cancer Research. Only those replicas which had a correlation score >0.9 were accepted for further analysis.