title: GSM8637824: Input, CYC, Testis, ChIP, REP#1; Drosophila melanogaster; ChIP-Seq
library_name: GSM8637824
library_construction_protocol: The ChIP protocol was modified based on Le Thomas et al. (2014). For each biological replicate, 200 heads, 200 pairs of testes, or 200 pairs of ovaries were collected at ZT10, fixed in 1% formaldehyde for 10 minutes, quenched with 25 mM glycine for 5 minutes, and washed three times with PBS. The fixed tissues were then homogenized in RIPA buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor, 10 mM NaF, 0.2 mM Na₃VO₄). Chromatin was sheared by sonication to fragments of 100–500 base pairs. The sample was then centrifuged at 13,000 × g for 10 minutes at 4°C, and the supernatant was collected. The supernatant was pre-cleared with Dynabeads Protein G beads (Invitrogen) for 2 hours at 4°C. 5% of the pre-cleared sample was reserved as the Input, while the remaining sample was incubated with 5 μL anti-GFP antibody (A-11122, Invitrogen) overnight at 4°C. The immunoprecipitated (IP) sample was then incubated with Dynabeads Protein G for 5 hours at 4°C to allow bead binding. After that, beads were washed 5 times in LiCl Wash Buffer (10 mM Tris pH7.4, 500 mM LiCl, 1% NP-40, 1% sodium deoxycholate), while the Input sample was incubated with 1 μL 10 mg/mL RNase A at 37°C for 1 hours. Rinsed in TE buffer (1 mM EDTA, 10 mM Tris–HCl, pH 8), and finally resuspended in Proteinase K buffer with 100 µg of Proteinase K. Finally, both samples were incubated with 100 μg of Proteinase K in PK Buffer (200 mM Tris, pH 7.4, 25 mM EDTA, 300 mM NaCl, 2% SDS) first at 55°C for 3 hours, followed by overnight incubation at 65°C. Illumina library preparation and sequencing were performed at the Novogene sequencing platform (https://cn.novogene.com/).
identifiers.namespace: GEO
identifiers.id: GSM8637824_r1