GSM7498458: WT H3K27me3 low salt rep 1; Homo sapiens; OTHER
CUT&RUN was performed according to the CUT&RUN Assay Kit protocol (Cell Signaling Technology) with the following modifications. Cells were detached with Accutase (Sigma). 200,000 cells were collected per experiment and pelleted by centrifugation for 5 minutes at 600 x g. Beads were incubated in primary antibody on a nutator overnight at 4°C. MNase digestion was carried out at 0°C in an ice water bath for 30 minutes. Salt fractionation and DNA purification were carried out according to the CUT&RUN.salt protocol (PMID: 29386331). Briefly, after STOP buffer addition, samples were incubated at 4°C for 1 hour to release low-salt fragments. Beads were resuspended in low salt buffer (175mM NaCl, 10mM EDTA, 2mM EGTA, 0.1% TritonX-100, 20μg/mL glycogen), rocked at 4°C for 1 hour, centrifuged for 5 minutes at 16,000 x g, and the supernatant containing the low salt fraction was collected. High salt buffer (825mM NaCl, 10mM EDTA, 2mM EGTA, 0.1% TritonX-100, 20μg/mL glycogen) was added to beads dropwise with gentle vortexing. Samples were again rocked at 4°C for 1 hour, centrifuged for 5 minutes at 16,000 x g, and the supernatant containing the high salt fraction was collected. Low salt fractions were adjusted to 500mM NaCl. RNAse A (Thermo Fisher Scientific) was added to all fractions and samples were incubated at 37°C for 20 minutes. SDS (sodium dodecyl sulfate) and Proteinase K (Cell Signaling Technology) were added and samples were incubated at 50°C for 1 hour. DNA was extracted using phenol/chloroform and precipitated in 100% ethanol as described. CUT&RUN libraries were constructed using the NEB Ultra II DNA library prep kit for Illumina with at least 12 cycles of PCR amplification. Libraries were profiled using the Agilent TapeStation D1000 high sensitivity ScreenTape on the Agilent 4150 TapeStation System