GSM5289853: lidKD_K27me3_Ov_rep2; Drosophila melanogaster; ChIP-Seq
Cut&Run in Drosophila tissues was previously described (Ahmad and Spens, 2019). Briefly, ovaries from 3-day-old flies were dissected in Wash+ Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.9 mM spermidine, 0.1% BSA with Roche cOmplete protease inhibitor) and were bound to BioMag Plus Concanavalin-A-conjugated magnetic beads (ConA beads, Polysciences, Inc). Tissue was then permeabilized for 10min in dbe+ Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.9 mM spermidine, 2 mM EDTA, 0.1% BSA, 0.05% digitonin with Roche cOmplete protease inhibitor). Samples were then incubated with gentle rocking overnight at 4°C with anti-H3K27me3 antibody solution (Cell Signalling Technology #9733, final concentration of 1:100 in dbe+ buffer). Protein A fused to micrococcal nuclease (p-AMNase) was added in dbe+ buffer and samples were incubated with rotation at room temperature for 1 hour. Cleavage was done in WashCa+ buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.9 mM spermidine, 0.1% BSA, 2 mM CaCl2 with Roche cOmplete protease inhibitor) at 0 ̊ for 30 minutes. Digestion was stopped with addition of 2XSTOP Buffer (200mM NaCl, 20mM EDTA, 4mM EGTA, 62.5µg/mL RNaseA). Samples were incubated at 37°C for 30 min to digest RNA and release DNA fragments. Cleaved DNA was then recovered with Ampure XP beads (Beckman Coulter) immediately after protease treatment. Cut&Run samples were purified using Agencourt AMPure XP beads (Beckman Coulter) and quantified with the Qubit (Invitrogen). Cut&Run-seq libraries were prepared from10 ng of double-stranded purified DNA using the MicroPlex Library Preparation kit v2 (C05010014, Diagenode s.a., Seraing, Belgium), according to manufacturer's instructions. In the first step, the DNA was repaired and yielded molecules with blunt ends. In the next step, stem-loop adaptors with blocked 5 prime ends were ligated to the 5 prime end of the genomic DNA, leaving a nick at the 3 prime end. The adaptors cannot ligate to each other and do not have single-strand tails, avoiding non-specific background. In the final step, the 3 prime ends of the genomic DNA were extended to complete library synthesis and Illumina compatible indexes were added through a PCR amplification (7cycles). Amplified libraries were purified and size-selected using Agencourt AMPure XP beads (Beckman Coulter) to remove unincorporated primers and other reagents.