CUT&RUN analysis for NOP16-mediated H3K27me3 alteration in MDA-MB231 cells [CUT&RUN-seq]

The goal of CUT&RUN-seq is to identify the global alteration of H3K27me3 levels by NOP16 overexpression or deletion in triple negative breast cancer cell line MDA-MB231 cells. Three (or Two) biological replicates were assigned for each group and in total 6 groups were prepared for these CUT&RUN-seq libraries. We mapped about 20 million reads per sample to hg38 human reference genome, and counted and normalized each reads number and identified H3K27me3 distribution. (1) CUT&RUN-seq analysis using MDA-MB231-NOP16 stably overexpression(OE1 or OE4) and empty vector control(E.V.) (2) CUT&RUN-seq analysis using MDA-MB231-NOP16 bulk KO (KO1 or KO4) and control line(no guide RNA control: n.g.) 1.5x105 nuclei extracted from each cell lines were used for CUT&RUN reaction (Skene, P. J. & Henikoff, S, eLife, 2017). CUT&RUN library were generated for NGS sequencing in biological triplicate (or partially duplicate), and sequenced by Illumina NextSeq 500.