title: GSM7061223: Input, WT, LBF, rep1; Drosophila melanogaster; RIP-Seq
library_construction_protocol: 0-12h embryos were collected, immediately frozen on dry ice, and stored at -80oC until RNA purification. The larval brain fraction (LBF), was dissected, immediately frozen on dry ice and stored at -80oC until RNA isolation. The RNA immunoprecipitation was performed essentially as described in Dominissi et al. Briefly, total RNA was isolated using RNeasy Maxi Kit (Qiagen). For each sample 1 mg of total RNA (1 μg/μl) was divided into batches of 45µg and incubated at 94°C in fragmentation buffer (100 mM Tris-HCl pH7.0, 100 mM ZnCl2) for 40 seconds. Fragmented RNA batches were pooled, and ethanol precipitated at -80˚C overnight. RNA samples were washed with 75% ethanol and resuspended in RNase-free water. Fragmentation efficiency was checked on a Bioanalyzer RNA chip (Agilent). RNA fragments were denatured by heating at 70°C for 5 minutes, then chilled on ice for 5 minutes. For immunoprecipitation, RNA samples were incubated overnight at 4°C with 12.5 µg of anti-5-hmC antibody (Diagenode rat monoclonal MAb-633HMC) or without antibody as negative control in IP buffer (750 mM NaCl, 50 mM Tris-HCl pH7.4, 0.5% IGEPAL CA-630, RNasin 400 U/ml and RVC 2 mM). 60 μl of equilibrated Dynabeads Protein G (Life Technologies) were added to the samples and incubated at 4°C for 2.5 hours.  The magnetic stand beads were washed with 1 ml IP buffer for 5 minutes three times. To elute immunoprecipitated RNA, 1 ml TriPure Reagent (Roche) was added, mixed thoroughly and centrifuged at room temperature for 5 minutes. Aqueous phase was recovered, and equal amount of chloroform was added, vortexed and aqueous phase was collected after centrifugation and ethanol precipitated at -80˚C overnight. RNA was resuspended in nuclease free water and used for library preparation. All buffers contained cOmplete EDTA-free protease inhibitor cocktail (Roche). Library preparation was done with the TruSeq ChIP Sample Prep Kit (Illumina) after reverse transcription of pulled-down RNA and synthesis of a second strand (NEB) by Next mRNA second strand synthesis module (NEB)). Briefly, 5 to 10 ng dsDNA was subjected to 5' and 3' protruding end repair. Then, non-templated adenines were added to the 3' ends of the blunted DNA fragments. This last step allows ligation of Illumina multiplex adapters. The DNA fragments were then size selected in order to remove all unligated adapters and to sequence 200-300-bp fragments. 18 cycles of PCR were carried out to amplify the library. DNA was quantified by fluorometry with the Qubit 2.0 and DNA integrity was assessed with a 2100 bioanalyzer (Agilent). 6 pM of DNA library spiked with .5% PhiX viral DNA was clustered on cBot (Illumina) and then sequenced on a HiScanSQ module (Illumina).