GSM8447936: C3H10T1-2_NSD1-2-3-SETD2-ASH1L-QuiKO_CUTandRUN_H3K27me3_rep2; Mus musculus; OTHER
CUT&RUN reactions were performed with the Epicypher CUTANA ChIC/CUT&RUN Kit Version 3 (cat# 14-1048) using 550,000 fresh C3H10T1/2 cultured cells and following the manufacturer's protocol (Epicypher User Manual Version 3.5) for adherent cells. Prior to performing CUT&RUN, optimization of C3H10T1/2 cell permeabilization was performed following Appendix 1.1 (Epicypher User Manual Version 3.5). Cell permeabilization was found to be most optimal using a final working concentration of 0.01% Digitonin (Promega). Successful binding of cells to Concanavalin A beads was confirmed as recommended and outlined by the manufacturer. Manufacturer supplied H3K4me3 positive control and IgG negative control antibodies were used and the reactions were performed in parallel with processed samples. Processed samples were incubated with specific antibodies. Manufacturer also supplied E.coli spike-in DNA, which was used in each reaction for sequencing normalization. Quantification of purified CUT&RUN DNA was performed using the Qubit fluorometer with the 1X dsDNA HS Assay Kit (Invitrogen). Library preparations were performed using the Epicypher CUTANA CUT&RUN Library Prep Kit Version 1 (cat# 14-1001) following the manufacturer's protocol. Final CUT&RUN library concentrations were quantified as previously described, and enrichment of mononucleosome-sized fragments was assessed by Bioanalyzer (Agilent) prior to sequencing. CUT&RUN libraries were sequenced at a depth of 15M reads per sample by the McGill University Genome Centre using the Illumina NovaSeq 6000 at 100bp paired-end reads.