GSM5661648: Drosophila Pupa-2hr-CPD_rep2; Drosophila melanogaster; OTHER
After repair, samples (embryo, larvae, pupae ,adults or s2 cells) were collected with ice-cold PBS and washed several times. 320 µl TE pH 8.0 was added to each sample and samples were ground. After grinding, 40 µl 10% SDS was added to each sample and samples were incubated at 70°C for 30 minutes. After incubation, 100 µl 5 M NaCl was added to each sample and they were incubated at 4°C overnight. After overnight incubation, samples from each developmental phase were centrifuged at high speed at 4 degress for one hour.  Supernatants were processed for excision or XR-Seq assay. Briefly, supernatants were incubated with 5 µL Rnase A and then 5 µL Proteinase K, purified, and then immunoprecipitated with anti-CPD, anti-6-4 (P-P) or anti-cisplatin antibodies.  Immunoprecipitates were then either radiolabeled and excision products were separated on 10-11% sequencing gels, or, for  XR-seq assay, immunoprecipitates were ligated to the adaptors, purified by a second immunoprecipitation, and DNA damages were reversed (using photoreactivation for photoproducts, and cyanide treatment for cisplatin damage). Libraries were generated by PCR and then sequenced.