GSM7070146: N1-M-LK-H3K9me3; Mus musculus; OTHER
Briefly, cells were collected 48 hours after rAAV transduction by trypsinization and washed with PBS. Nuclei were extracted with NE buffer. Nuclei were quantitated after Trypan Blue staining using an automated cell counter (Countess, Thermo Fisher) and diluted accordingly. 1e5 nuclei per target protein were used for Cut&Tag. Nuclei were mixed with activated Concanavalin A beads. After successive incubations with primary antibody (overnight) and secondary antibody (for 30 min) in antibody and Digitonin150 buffer respectively, the beads were washed with Digitonin150 buffer and resuspended in Digitonin300 buffer with 2.5uL of pA(G)-Tn5 for 1 hr and washed with the same buffer. Incubations were performed at room temperature in low-retention PCR strip tubes (Epicypher). Tagmentation was performed for 1 hr at 37C in Tagmentation buffer that provides MgCl2. Beads were washed with TAPS buffer and DNA material was released by adding SDS Release Buffer and incubating at 58C for 1hr. Quenching of SDS was performed by adding SDS Quench buffer and PCR was performed directly on this material. Universal P5 and indexed P7 primer solutions were used (see Supplemental Table 3 for sequences), and 21 cycles of PCR were performed. Clean-up was performed with AMPure beads, eluted in 15uL 0.1x TE buffer. Qubit and Bioanalyzer were used to verify library qualities before pooling samples for sequencing. The barcoded libraries were mixed to achieve equimolar representation aiming for a 10nM final concentration. Cut & Tag. Sequencing was performed with Illumina HiSeq 4000, 150 cycles total per lane, 2x75 paired-end reads, depth of >15M reads per sample.