NextSeq 500 sequencing; Identification of RNAs associated with the Caenorhabditis elegans SL1 snRNP SNA-1 protein by RIP-Seq
Identification of RNAs associated with the Caenorhabditis elegans SL1 snRNP SNA-1 protein by RIP-Seq
C. elegans embryo extracts were prepared essentially as described (Lasda, Kuersten, and Blumenthal 2011).  Briefly, synchronised populations were prepared by alkaline hypochlorite treatment of gravid adult animals grown on NGM agar plates in the presence of OP50 bacteria, or in liquid culture in S-complete medium supplemented with OP50. Approximately 2x10^5 embryos were transferred into 500 ml S-complete medium (50 mM potassium phosphate buffer (pH 6), 100 mM NaCl, 10 mM potassium citrate (pH 6), 3mM CaCl2, 3mM MgSO4 and 1x standard trace metal solution) supplemented with Nystatin (14 mg/ml) and grown overnight at 20°C in a shaking incubator at 160 rpm to L1 stage. After approximately 24 h, 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 and twice 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 1873560) and 1mM PMSF) and finally resuspended in 1 volume homogenisation buffer.  They were lysed using a Dura-Grind Dounce Tissue Grinder (Wheaton) and the extract was then cleared by centrifugation at 10,000 × g for 10 min at 4°C.  The supernatants was dialysed against 20 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM DTT, 10% glycerol, 0.5 mM EDTA, 1 mM PMSF. Total RNA for control samples was isolated using the MagMAX™ mirVana™ Total RNA Isolation Kit (ThermoFisher) following manufacturers instructions.  Libraries were prepared using the Diagenode CATS small RNA sequencing following the manufacturer's instructions