GSM9571790: Pzg-GFP ovary Input 2; Drosophila melanogaster; ChIP-Seq
100 ovaries from 3 to 5 days old Pzg-GFP females were prepared in PBS. Crosslinking was performed in 500 µl of 1% formaldehyde for exactly 10 min at RT, which was stopped by adding 500 µl 50 mM glycine. After five minutes of gentle rotation at RT, the samples were placed on ice. The ovaries were then washed three times with PBS and twice with Farnham buffer [5 mM HEPES pH 8.0, 85 mM KCl, 0.5 % NP-40, 1 tablet of protease inhibitor cocktail, 10 mM NaF, 0.2 mM Na3VO4 in ddH2O] for 5 minutes each at 4°C. The ovaries were then homogenised in 100 µl RIPA-2 buffer [20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 % sodium deoxycholate (SAC), 0.1 % SDS, 1 tablet of protease inhibitor cocktail, 10 mM NaF, 0.2 mM Na3VO4 in ddH2O] in a glass homogenizer with a size B pestle on ice and sonicated in 12 cycles (30s on, 90s off) with a Branson Sonifier Cell Disruptor B15. After centrifugation at 20.000 x g for 10 min at 4°C, 100 µl of lysate per IP were used and 5% input control was removed from each lysate. Incubation with 10 µg rabbit ⍺-GFP antibody was carried out overnight at 4°C. The following day, the lysate-antibody mixture was added to 50 µl of Dynabeads™ Protein G and incubated for 5 hours at 4°C. While the Dynabeads™ were washed five times for 10 minutes at 4°C, the input control was digested for 1 hour at 37°C with 1 µl 10 mg/ml RNase. After adding 713 µl PK buffer [200 mM TRIS-HCl pH 7.4, 25 mM EDTA, 300 mM NaCl, 2 % SDS, 100 µg Proteinase K] and 277 µl ddH2O to the input and 713 µl PK buffer and 267 µl ddH2O to the Dynabeads™, the samples were incubated for 3 hours in a 55°C water bath. Reverse crosslinking was performed overnight in an oven at 65°C. The following day, the DNA was extracted using phenol-chloroform, precipitated with ethanol and dissolved in 10 µl TE. All samples were sequenced using Illumina's NextSeq 500 System and NextSeq 500/550 Mid-Output v2.5 Kit, generating single-end reads with a length of 150 bp.