GSM7110168: S2-Grh_DMSO_IgG_rep2; Drosophila melanogaster; OTHER
CUT&RUN was performed using EpiCypher reagents according to the manufacturer protocol. 2×105 cells were used for each CUT&RUN reaction. Overnight incubation with antibodies was performed in antibody buffer containing 0.05% digitonin. 0.5 µL anti-H3K27me3 (Cell signaling technologies) or rabbit IgG were used for CUT&RUN. Prior to addition of antibody, a 1:5 dilution of K-MetStat Panel spike-in nucleosomes (EpiCypher, cat. # 19-1002) was added to each reaction. Libraries were prepared using the NEBNext Ultra II kit (NEB). During library preparation, cleanup steps were performed using a 1.1X ratio of Axygen paramagnetic beads, as recommended by EpiCypher. PCR amplification was performed with the following conditions: 98°C for 45 seconds, then 14 cycles of 98°C for 15 seconds, 60°C for 10 seconds, and a final extension of 72°C for 1 minute.