Binding site analysis and identification of reciprocal targeting of Dom-A and Xbp1 in Drosophila melanogaster
Binding site analysis and identification of reciprocal targeting of Dom-A and Xbp1 in Drosophila melanogaster
CUT&RUN was performed as previously described (Meers M.P. et.al., 2019. DOI: 10.7554/eLife.46314). For each condition, 10⁶ cells were washed three times with Wash Buffer (WB: 20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, cOmplete EDTA-free Protease inhibitor), centrifuging at 600 x g for 3 min. Cells were resuspended in 1 mL WB per antibody. Concanavalin A beads (10 μL/sample) were activated by two washes in Binding Buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2), mixed with cell suspension and incubated for 10 min at RT with rotation. Beads were collected on a magnet and resuspended in 150 μL Antibody Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.05% digitonin, 2 mM EDTA, 0.5 mM Na-butyrate) containing diluted antibodies, followed by incubation for 2 h at RT or overnight at 4°C with slow rotation. After two washes with Dig-Wash Buffer (DWB: 20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.05% digitonin, cOmplete EDTA-free Protease inhibitor), beads were resuspended in 150 μL pAG-MNase solution (700 ng/mL in DWB) and incubated at 4°C for 1 h with slow rotation. After two further washes, beads were resuspended in 100 μL DWB, chilled, and digestion was initiated with 2 μL 100 mM CaCl2 for 30 min. Reactions were stopped with 100 μL 2x Stop Solution (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% digitonin, 100 μg/mL RNase A, 50 μg/mL glycogen) and incubated at 37°C for 30 min to release chromatin. Supernatants were collected on a magnet. Reverse crosslinking was done with 2 μL 10% SDS and 2.5 μL Proteinase K (20 mg/mL) at 50°C for 1 h. DREX-ChIP was performed in chromatinized genomic DNA. Briefly, Drosophila embryonic extract from the pre-blastoderm stage was incubated with DNA in the presence of ATP. Samples were mildly fixed with 0.1% formaldehyde for 10 min, quenched with 125 mM glycine for 5-10 min, and fragmented by 3 min MNase digestion to yield mononucleosomes. Reactions were adjusted to 1 mL with RIPA Buffer (25 mM HEPES-NaOH pH 7.6, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 0.1% sodium deoxycholate, freshly added 1 mM DTT, cOmplete EDTA-free Protease inhibitor). Protein A/G beads (20 μL slurry; Cytiva, Cat. Nos. 17-0618-05, 17-5280-05) were equilibrated in RIPA (3 x 5 min washes). For pre-clearing, samples were incubated with beads for 1 h at 4°C, then beads were removed. Antibody (1 μL) was added and incubated overnight at 4°C with rotation. The following day, 20 μL equilibrated beads were added for 3 h at 4°C, then washed three times with RIPA. DNA was eluted by incubating beads in 100 μL 1x TE with 3.2 μL 10% SDS and 10 μL Proteinase K (10 mg/mL) at 56°C overnight. Cells were cultured at 26°C in Schneider's Drosophila Medium (Thermo-Fischer, Cat. No. 21720024) supplemented with 10% FBS (PAN Biotech, Cat. No. P30-3031) and 1% Penicillin-Streptomycin solution (PenStrep; Sigma-Aldrich, Cat. No. P-4333). Double-stranded RNA (dsRNA) targeting the desired sequences was generated by in vitro transcription using the HiScribe T7 High Yield RNA Synthesis Kit (NEB, Cat. No. E2040S). For CUT&RUN, the cells underwent two consecutive rounds of 6 μg dsRNA for a total of 7 days. Non-targeting dsRNA (GST) was included in the RNAi experiments as a negative control. For Xbp1 CUT&RUN cells were treated without or with 5 mM DTT for 1 hour prior to CUT&RUN.  DNA was purified by phenol-chloroform extraction, followed by ethanol precipitation (2 mg/mL glycogen, 500 μL 100% ethanol, 15 min on ice). Pellets were washed with 100% ethanol and resuspended in 30 μL 0.1x TE buffer. For CUT&RUN a modified version of NEBNext Ultra II DNA Library Prep Kit (NEB, Cat. No. E7645S) protocol was followed as described in (Kong et al., 2021. DOI: 10.1016/j.xpro.2021.100750 ).  For ChIP, manufacturer`s protocol was carried out.