Chromatin immunoprecipitation (X-ChIP) was prepared as describe previously (Erokhin et al., BMC Biol, 2021). For each experiment, 150-200 mg of 3rd instar larvae or embryos were collected. The material was homogenized in 5 ml of buffer A1 (15 mM HEPES, pH 7.6; 60 mM KCl, 15 mM NaCl, 4 mM MgCl2, 0.5% Triton X-100, 0.5 mM DTT, Roche cOmplete protease inhibitor) supplemented with the EDTA-free protease inhibitor cocktail (Roche, Switzerland) and formaldehyde as a crosslinking agent (final concentration 1.8%). The reaction was stopped by adding glycine (final concentration 225 mM). The homogenate was cleared by passing through 100-µm nylon cell strainer (BD Falcon) and pelleted by centrifugation at 4000 g, 4°C for 5 min. After twice washing in buffer A1 at 4°C, the pellet was treated with 0.5 ml of complete lysis buffer (15 mM HEPES, pH 7.6; 140 mM NaCl, 1mM EDTA, 0.5 mM EGTA, 1%Triton X-100, 0.5 mM DTT, 0.1% sodium deoxycholate, 0.1% SDS, 0.5 % N-lauroylsarcosine, Roche cOmplete protease inhibitor) and sonicated to break chromatin into fragments with an average length of 200-600 bp. The material was pelleted by centrifugation at 18 000 g for 5 min, and the supernatant fluid was transferred to a new tube. The pellet was treated with the second 0.5-ml portion of lysis buffer, and the preparation was centrifuged at 18 000 g for 5 min. The two portions of the supernatant fluid were pooled, cleared by centrifuging twice at 18 000 g for 10 min, and the resultant chromatin extract (1 ml) was used for ChIP experiments. 9 ml of ChIP dilution buffer (15 mM HEPES, pH 7.6; 140 mM NaCl, 1mM EDTA, 0.5 mM EGTA, 1%Triton X-100, 0.5 mM DTT, Roche cOmplete protease inhibitor), was added to the sheared chromatin together with 50 ul of prewashed Protein A Sepharose beads (GE Healthcare). The samples were incubated at 4 oC for 1 hour on a rotating platform. The samples were spun at 10,000g to remove the beads and the supernatant was transferred to a fresh tube. One aliquot (1/10 volume) of chromatin extract after preincubation with Sepharose was kept as a control sample (Input). DNA concentration of 5ul was measured using a Qubit 3.0 fluorometer (Invitrogen). 1.5ng of starting material/sample was used to make each library. ChIP-seq libraries were obtained using the NEBNext Ultra™ II DNA library preparation kit (New England Biolabs) or with the Thruplex DNA-seq and single index kits (Takara) following the manufacturer's instructions. Samples were sequenced by 50bp or 100bp single-end sequencing with HiSeq2500 (Illumina) or with NovaSeq6000 sequencer.