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. Immunoprecipitations of GFP-tagged proteins were done in triplicates using anti-GFP nanobody coupled agarose beads and control agarose beads (GFP-Trap and control agarose beads, Chromotek GmbH). For immunoprecipitation, extract supplemented with 35 mM KCl, 4 mM MgCl2, 1.5 mM DTT, 3% PEG 8000 was mixed with settled anti-GFP nanobody coupled agarose beads or control agarose beads and incubated at 4°C for 1h. Then the beads were collected by centrifugation and washed extensively with 20 mM Tris-HCl (pH8.0), 100 mM KCl, 0.1 mM EDTA, 0.1% NP-40, 1 mM DTT. After removal of the final wash, the beads were treated with 200 µl 0.2 mg/ml Proteinase K in 200 mM Tris-HCl (pH7.5), 300 mM NaCl, 2% SDS and 25 mM EDTA at 65°C for 15 min. Then RNA was recovered by two subsequent extractions with acid-phenol:chloroform (pH 4.5), and concentrated by ethanol precipitation using linear acrylamide as carrier, washed with 80% ethanol and air dried. Libraries were prepared using the Diagenode CATS small RNA sequencing following the manufacturer's instructions