GSM9365425: Mio GKD, h3k9me3 mark, rep 1; Drosophila melanogaster; OTHER
Ovaries were dissected in PBS. PBS was removed and the samples were permeabilized in 1mL of Permeabilization Solution (PBST+1% Triton-X) rotating in RT for 1 hour. Samples were then incubated overnight at 4°C in primary antibody dilutions in freshly prepared BBT+ buffer (PBST + 1% BSA + 0.5 mM Spermidine + 2 mM EDTA + 1 large Roche complete EDTA-free tablets). Primary antibody was replaced with BBT+ buffer and quickly washed twice. Samples were then incubated in ~700 ng/ml of pAG-MNase in BBT+ buffer rotating for 4 hours at 25°C. Samples were then quickly washed twice in wash+ buffer (20 mM HEPES pH7.5 + 150 mM NaCl + 0.1% BSA + 0.5 mM Spermidine + 1 large Roche complete EDTA-free tablets in water). Samples were resuspended in 150 μl Wash+C (wash+ + 100 mM CaCl2) and incubated for 45 minutes on nutator at 4°C. The cleavage reaction was terminated by addition of 150 μl StopR (NaCl final 200 mM + EDTA final 20 mM + 100μg/mL RNaseA) and incubating the sample at 37˚C for 30 minutes. Samples were then centrifuged at 16,000 x g for 5 minutes and 300 μl of the supernatant was collected for DNA discovery. To the supernatant, 2 μL 10% SDS and 2.5 μL of 20 mg /mL Proteinase K was added and incubated at 50°C for 2 hours. Half of this was kept as a backup and half was used in bead cleanup. 20 μL AmpureXP bead slurry and 280 μL MXP buffer (20% PEG8000 + 2.5 M NaCl + 10 mM MgCl2 in water) was added to the sample and mixed thoroughly followed by 15 minutes incubation at RT. The beads were separated by magnet and supernatant was discarded. The beads were carefully washed with 80% ethanol for 30 seconds, while on the magnetic stand and air dried for 2 minutes. The beads were then resuspended in 10 μL DNase free water. The samples from CUT&RUN assay were used for library preparation using NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (E7645, E7103) and adaptor ligated DNA were prepared without size selection.