GSM6294370: Head input, standard conditions, rep 3; Drosophila melanogaster; RIP-Seq
800 fly heads were isolated from bodies by sieving for CLIP. All samples were stored at -80 C. Phenol-chloroform (Invitrogen 15596018) and RIPA buffer (150 mm NaCl, 1% Nonidet P-40, 0.5% Sodium deoxycholate, 0.1% SDS, 50mM Tris pH 7.5) were added to frozen samples and homogenized by bead bashing (Bead Ruptor, 19-040E). Phenol-chloroform extracted RNA was precipitated by isopropanol with GlycoBlue coprecipitant (Invitrogen AM9515). RNA was stored at -80C until further processing. 20 ug of poly(A) selected (Invitrogen, 61012) RNA for each biological replicate from heads (~800, W1118cs) of flies was used as input to the CLIP reaction. Fragmented RNA (Ambion E6150S) was incubated for 2 hr at 4 C with antibody against m6A (Abcam, ab151230), UV-crosslinked, then immunoprecipated with magnetic beads (Invitrogen, 10004D). Pre-adenylated dye-conjugated linkers were ligated (NEB M0351S) to dephysophorylated 3' ends of RNA fragments overnight at 16 C. RNA was extracted with NuPAGE SDS buffer and separated by NuPAGE gel (Invitrogen, NP0321), transferred to nitrocellulose membrane and visualized for excision at 800 nm. RNA-antibody complexes were released from the membrane by proteinase K digestion and RNA was purified with phenol chloroform extraction. RNA was reverse transcribed (Invitrogen, 18080085), circulized (Lucigen, CL4111K), PCR-amplified (Thermo Scientific, F530S). Libraries were subjected to 151bp paired-end sequencing according to the manufacturer's protocol on an Illumina NovaSeq 6000 at the University of Michigan sequencing core.  CLIP-seq