title: GSM6048125: Kc167_mock_CP190_R2; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: Total of 2–3 × 107 cells were fixed with 1% formaldehyde and chromatin was prepared as described previously (Bag et al., 2021). 300 µg of chromatin was diluted to 1:5 with IP dilution buffer and incubated with rabbit normal serum for 1h on rotator at 4 °C. Then precleared chromatin was collected by centrifugation at 2000g for 5 min at 4 °C. Chromatin was added to 100 µl prewashed Protein A Sepharose beads (cytiva) and 50 µL of rabbit anti-serum against Isha and rotated overnight at 4°C. The next day beads were washed with the following wash buffers: two times with low-salt wash buffer (20 mM Tris-HCl pH 8, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% SDS), two times with high-salt wash buffer (20 mM Tris-HCl pH 8, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% SDS), and once with LiCl wash buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1% deoxycholate). Chromatin was eluted two times with 200 μl of elution buffer (0.1 M NaHCO3, 1% SDS) for 30 min at 65°C each in a thermomixer at 800 rpm. Then chromatin was de-crosslinked with solution (20 μl of 5 M NaCl, 8 μl of 0.5 M EDTA, 10 μl of 1 M Tris-HCl pH 8) by incubating overnight at 65°C. After de-crosslinking, elutes were treated with 4 µl of proteinase K (20 mg/ml) for 2 h at 50°C and further purified using phenol-chloroform followed by ethanol precipitation with 0.1 vol of 3 M NaOAc pH 5.2 and 2.5 vol of 100% ethanol supplemented with 2 μl of Glycoblue (Ambion) and incubated overnight at −80°C. Next day, samples were centrifuged at 10,000 g for 20 min at 4°C and pellets were washed with 70% ethanol. Pellets were air-dried at room temperature and then resuspended with 10 μl of nuclease-free water. For Chromatin immunoprecipitation with guinea pig anti-serum against Su(Hw), rabbit anti-serum against CP190, rabbit anti-serum against Mod(mdg4)67.2 followed the same methods as mentioned earlier (Bag et al., 2021). ChIP-seq library was performed as described previously (Bag et al., 2021). All samples were sequenced with HiSeq3000 (Illumina) using 50 bp single-end sequencing.