title: GSM6048138: NE_input_R3; Drosophila melanogaster; RIP-Seq
library_construction_protocol: Drosophila nuclear extract was prepared from 30 g of mixed stage (0–24 h) embryos as described previously (Bag et al., 2021) and nuclei were lysed with HBSMT buffer, supplemented with 1 U/μl RNasin (Promega). 12 mg nuclear extract was used per IP. Nuclear extracts were incubated with 600 μl Protein A conjugated sepharose (cytiva) prebound to 300 μl preimmune rabbit serum on rotator for 1 h at 4°C. Then precleared extracts were then transferred to 600 μl Protein A sepharose prebound to 300 μl rabbit anti-serum against Isha and incubated rotating for 1 h at 4°C. Next, beads were washed in HBSMT three times and HBSM twice. Bound RNA was isolated directly from Protein A beads by acid Phenol:Chloroform (Thermo Fisher Scientific) extraction followed by ethanol precipitated with 3 M NaOAc. RNA was DNase I (Roche) treated and purified further using acid Phenol:Chloroform extraction and ethanol precipitation. Then RNA was chemically fragmented using RNA Fragmentation Reagents (Thermo Fisher Scientific) and purified by ethanol precipitation with 3 M NaOAc. 100 ng of RNA was used for each RNA-seq library preparation using Ovation RNA-seq Systems 1–16 for Model Organisms (Nugen) according to manufacturer's instruction. Then samples were sequenced on HiSeq3000 (Illumina) using 50 bp single-end sequencing at the NIDDK Genomics Core Facility.