title: GSM6165872: H3K9me2 Kdm3 GLKD biol rep 1; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: ChIP-seq: Chromatin immunoprecipitation (ChIP) was performed as described in Lee et al., 2006. Briefly, 100 ovary pairs were manually dissected into Schneider media and cross-linked in 1% formaldehyde/PBS for 10 min at room temperature with agitation. The cross-linking reaction was quenched by STOP buffer (PBS 1X, Triton 0.1%, Glycine 1 M) and ovaries were washed in PBS and homogenized in a glass douncer: first slightly dounced in PBST 0.1% and centrifugated 1 min 400g, followed by strong douncing in cell lysis buffer buffer (KCL 85 mM, HEPES 5 mM, NP-40 0.5% , Sodium butyrate 10 mM, EDTA free protease inhibitor cocktail Sigma) following by 5 min centrifugation at 2000g. We performed 2 washes with cell lysis buffer. The homogenates were then lysed on ice for 30 min in nucleus lysis buffer (HEPES 50 mM, EDTA 10 mM, N lauryl sarkosyl 0.5%, sodium butyrate 10 mM, EDTA free protease inhibitor cocktail Sigma). DNA was sheared using a Bioruptor pico from Diagenode for 10 cycles (30 sec on, 30 sec off). The sonicated lysates were cleared by centrifugation and then incubated overnight at 4°C with anti-Rhino antibody. Then 40 µL of Protein A Dynabeads was then added and allowed to bind antibody complexes by incubation for 1 h at 4°C. For H3K9me2 and me3 ChIP, 50 µL of Protein A Dynabeads were first coated with the anti-H3K9me3 or me2 antibodies and then incubated with the chromatin overnight at 4°C. Following four washing steps with high salt buffer (Tris pH 7.5 50 mM, NaCl 500 mM, Triton 0.25% , NP-40 0.5% , BSA 0.5% , EDTA pH 7.5 5 mM), DNA-protein complexes were eluted and de-cross-linked 10 h at 65°C. RNA and protein was digested by RNase A and Proteinase K treatments, respectively, before purification using Phenol/Chloroform protocol. Barcoded libraries were prepared using Illumina technology