Identification of RNAs associated with the Caenorhabditis elegans spliced-leader trans-splicing proteins SNA-2, SNA-3, SNR-2 and SUT-1 proteins by RIP-Seq.
Identification of RNAs associated with the Caenorhabditis elegans spliced-leader trans-splicing proteins SNA-2, SNA-3, SNR-2 and SUT-1 proteins by RIP-Seq.
Embryonic extracts from C. elegans strains PE912, PE918, PE975 and PE1038 expressing GFP-tagged SUT-1, SNR-2, SNA-3 or SNA-2 proteins were prepared essentially as described (Fasimoye et al, 2022; https://doi.org/10.1093/nar/gkac534), with minor modifications. Synchronised populations were grown in liquid culture in S-complete medium supplemented with OP50. Approximately 1.5x10E6 embryos were transferred into a 2 L flask containing 500 ml S-complete medium (50 mM potassium phosphate buffer (pH 6), 100 mM NaCl, 10 mM potassium citrate (pH 6), 3 mM CaCl2, 3 mM MgSO4 and 1x standard trace metal solution) and grown overnight at 20°C in a shaking incubator at 200 rpm to L1 stage. After hatching of the embryos, 14-16 g frozen OP50 bacteria were added and the incubation was continued until synchronous gravid adult worms emerged. Adults were recovered by centrifugation at 400x g for 2 min, washed using MilliQ water, floated on 1 M sucrose and then washed again using MilliQ water to remove debris. Animals were then lysed by alkaline hypochlorite treatment and embryos recovered by floating on sucrose. The embryos were washed three times with MilliQ water, twice in 10mM Tris-HCl (pH 8.0), 1.5 mM MgCl2, 10 mM KCl, 50 mM sucrose, 0.05% NP-40,  and once in homogenisation buffer (10mM Tris-HCl (pH 8.0), 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, 50 mM sucrose, 0.05% NP-40, 1X cOmplete EDTA-free protease inhibitor (Roche) and 1mM PMSF) and finally resuspended in 3 volumes homogenisation buffer. Embryos were lysed using a Dura-Grind Dounce Tissue Grinder (Wheaton) and extracts were then cleared by 10 min centrifugation at 10,000 × g at 4°C followed by two times 5 min centrifugation at 10,000 × g at 4°C. The final supernatants were dialysed against 20 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM DTT, 10% glycerol, 0.5 mM EDTA, 1 mM PMSF at 4°C, and then flash-frozen in liquid nitrogen for storage at -80°C in an ultra-low temperature freezer.  Immunoprecipitations were done in quadruplicates as described (Fasimoye et al, 2022; https://doi.org/10.1093/nar/gkac534) using either anti-GFP nanobody agarose beads (GFP) or control agarose beads (control) (ChromoTek) and 500 µl extract supplemented with ¼ volume of 175 mM KCl, 20 mM MgCl2. and 20 µl settled agarose beads. After 1 h, the beads were collected by centrifugation at 2000x g for 2 min at 4°C and washed twice by resuspension in 500 µl of 20 mM Tris-HCl (pH8.0), 100 mM KCl, 0.1 mM EDTA, 0.1% NP-40, 1 mM DTT followed by centrifugation at 1000x g for 1 min at 4°C. Then they were transferred into fresh 1.5 ml tubes and washed 3 more times with the same buffer. After removal of the last wash, settled beads were treated with 200 µl 0.2 mg/ml Proteinase K in 200 mM Tris-HCl (pH7.5), 1% SDS and 1 mM EDTA at 65°C for 15 min. Then the RNA was recovered by two subsequent extractions with acidic phenol:chloroform:isoamylalcohol (125:24:1, pH 4.5), supplemented with 10 µg linear acrylamide and concentrated by ethanol precipitation, washed with 70% ethanol and air dried. Libraries were prepared using the Diagenode D-Plex Small RNA-seq Kit for Illumina following the manufacturer's instructions.