GSM9089739: Tip60 CUT&RUN, Tip60 RNAi, PPI-Serum (pre-immune serum) for CUT&RUN, Replicate 3; Drosophila melanogaster; OTHER
CUT&RUN was performed as previously described (Meers M.P. et.al., 2019. DOI: 10.7554/eLife.46314). Briefly, Concanavalin-A magnetic beads (10 μl of slurry/reaction) were pre-activated by washing 2 times with Binding Buffer (BB: 20 mM Hepes pH 7.5 (NaOH), 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2). 106 Kc167 cells per condition were bound to the beads, and washed twice with Wash Buffer (WB: 20 mM Hepes pH 7.5 (NaOH), 150 mM NaCl, 0.5 mM spermidine, 1x cOmplete EDTA-free Protease inhibitor). Cells were permeabilized using 150 μl of Antibody Buffer (AB: 20 mM Hepes, pH 7.5 (NaOH), 150 mM NaCl, 0.5 mM spermidine, 0.05% digitonin, 2 mM EDTA, 0.5 mM Na-butyrate). Antibodies were mixed into AB at the indicated dilutions. Samples were put on a nutator at RT for 2 hours, and washed twice with Dig-Wash Buffer (Dig-WB: 20 mM Hepes, pH 7.5 (NaOH), 150 mM NaCl, 0.5 mM spermidine, 0.05% digitonin, 1x cOmplete EDTA-free Protease inhibitor). pAG/MNase protein (700 ng/ml) was mixed into 150 μl of Dig-WB and samples were placed on a nutator at 4°C for 1 hour, and then washed twice with Dig-WB. Chromatin digestion was performed by the addition of 2 mM (f.c.) CaCl2 into 100 μl of Dig-WB and incubated at 4°C for 30 min. The reaction was stopped by adding 100 μl of 2x STOP buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% digitonin, 100 μg/ml RNase A, 50 μg/ml glycogen) and the chromatin fragments were released from insoluble nuclear chromatin by incubating at 37°C for 30 min. Samples were collected by a quick spin and placed on a magnet stand to concentrate the magnetic beads with bound chromatin fragments. Chromatin was digested with 0.25 mg/ml Proteinase K/ 0.1% SDS, and DNA was purified by phenol/chloroform extraction. DNA concentrations were determined using Qubit (Thermo Fisher).                                    ChIP-seq was performed using 70 million S2 cells fixed with 2.2 ml 10x Fixing Solution (0.1 M NaCl, 50 mM Hepes pH 7.6, 1 mM EDTA, 0.5 mM EGTA, 10% methanol-free formaldehyde) for 8 min at RT. Fixation was quenched with 125 mM glycine for 10 min on ice, cells were pelleted (1500 rpm, 10 min, 4°C), and washed twice with 10 ml ice-cold PBS. Nuclei were isolated in 1 ml 0.5% Triton/PBS supplemented with 1x cOmplete EDTA-free protease inhibitors and 2 mM Na-butyrate (rotate 15 min, 4°C), centrifuged (3000 rpm, 10 min), and washed once with PBS. Pellets were resuspended in 1 ml RIPA buffer (10 mM Tris pH 8.0, 140 mM NaCl, 1 mM EDTA, 0.1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100) supplemented with 2 mM CaCl2, 1x cOmplete EDTA-free protease inhibitors, 1 mM PMSF, and 2 mM Na-butyrate. Lysates of 1 ml were digested with 0.6 U MNase (Sigma–Aldrich), was resuspended in EX-50 (50 mM KCl, 10 mM HEPES, pH 7.6, 1.5 mM MgCl2, 0.5 mM EGTA, 10% glycerol) at 0.6 U/μl, at 37°C for 35 min and stopped with 22 μl 0.5 M EGTA on ice. Chromatin was mildly sheared with a Covaris AFA S220 (50 W peak power, 200 cycles/burst, 20% duty factor, 8 min, 4°C), centrifuged (16,000 g, 20 min, 4°C), and supernatant was pre-cleared with 10 μl Protein A+G beads per 100 μl chromatin (1 h, 4°C). Beads were removed (3000 rpm, 3 min), and 50 μl input was collected. For IP, 200 μl chromatin were diluted to 500 μl in RIPA, incubated overnight at 4°C with antibody or IgG control, and captured with 40 μl Protein A+G beads (3 h, 4°C). Beads were washed 5× with 1 ml RIPA, resuspended in 100 μl TE (10 mM Tris pH 8.0, 1 mM EDTA), and inputs adjusted to equal volume. Decrosslinking was performed with RNase A (50 μg/ml, 30 min, 37°C), followed by Proteinase K (0.5 mg/ml) and SDS (0.6%) at 68°C for 2 h. DNA was purified using AMPure XP beads. To 100 μl sample, 200 μl beads (1:1 dilution) were added, mixed by pipetting, and incubated 5 min at RT. Tubes were placed on a magnetic stand for ≥2 min, supernatant removed, and beads washed twice with 200 μl 80% ethanol. After a brief spin, residual ethanol was removed on the magnetic stand and beads were air-dried for 2 min. DNA was eluted in 30 μl TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0) by pipetting, incubated 2 min at RT, and collected after 1 min on the magnetic stand. For ChIP-seq samples, libraries were prepared using NEB Next Ultra II DNA Library (New England Biolabs) according to the manufacturer's instructions. For CUT&RUN, sequencing libraries were prepared as described (25). All libraries were sequenced on an Illumina NextSeq1000/2000 sequencer at the Laboratory of Functional Genomic Analysis (LAFUGA, Gene Center Munich, LMU). For CUT&RUN about 5x106 paired-end reads were sequenced per sample for each of the biological replicates.