title: iCLIP-seq identifies direct binding of PETISCO complex to replication-dependent histone mRNAs in Caenorhabditis elegans embryos
design: iCLIP-seq identifies direct binding of PETISCO complex to replication-dependent histone mRNAs in Caenorhabditis elegans embryos
library_construction_protocol: Wild-type worms were grown on egg plates and embryos harvested by bleaching. Embryos were UV cross-linked four times at 100 mJ/cm² (254 nm) in Worm Lysis Buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM DTT, 0.1% Triton X-100) and snap-frozen as \"worm balls.\" DYNAL™ Dynabeads™ Protein G (100 μl) were conjugated with 2 μg TOFU-6 antibody, or no antibody, for 1 h at room temperature and washed twice. Embryo extracts (1 mg total protein per replicate) were treated with RNase I and TURBO DNase at 37°C for 3 min, clarified by centrifugation, and filtered. Lysates were incubated with antibody-conjugated beads, or no-conjugated beads, for 2 h at 4°C, washed, and treated with T4 PNK for RNA 3'-end dephosphorylation. L3-App adapter ligation was performed overnight at 16°C, followed by RNA 5'-end labelling with ³²P-γ-ATP (37°C, 5 min). Samples were run on 4-12% NuPAGE Bis-Tris gels, transferred to nitrocellulose membrane, and visualized by phosphoimaging.  RNA-protein complexes were isolated from the nitrocellulose membrane, treated with Proteinase K, and RNA extracted using phenol/chloroform.  After precipitation, reverse transcription was performed using SuperScript III, followed by second adapter ligation and cDNA amplification (18-24 cycles). Libraries were assessed using TapeStation and quantified by Qubit. 
identifiers.namespace: European Bioinformatics Institute
identifiers.id: E-MTAB-15713:TOFU6_rep3_s