RNAse III-deficient HT115 bacteria were transformed with an L4440-derived plasmid, corresponding to the required RNAi treatment. A 0.5ml pre-culture was then grown overnight, and used to inoculate a 400 ml culture in ‘Terrific Broth’ (12 g/l Tryptone, 24 g/l yeast extract, 9.4 g/l K2HPO4, 2.2 g/l KH2PO4, adjusted to pH 7). After 7 hours of growth in a baffled flask at 37°C with agitation, expression of dsRNA was induced overnight at 20°C by addition of 3mM IPTG. The bacteria were then pelleted and resuspended with one-fifth volume of buffer (M9 medium supplemented with 75 mg/l cholesterol; 100 mg/l ampicillin; 50 mg/l tetracycline; 12.5 mg/l amphotericin B; 3 mM IPTG).
For each experiment, 1ml of a synchronized population of L4 worms expressing GFP-PSF-1 were fed for 50 hours at 20°C on a 15 cm RNAi plate (see above), supplemented with 8 g of bacterial pellet for the required RNAi treatment, prepared as described above. After feeding, the adults worms were washed in M9 medium and resuspended for 2 minutes at room temperature in 14 ml of ‘bleaching solution’ (for 100 ml: 36.5 ml H2O, 45.5 ml 2N NaOH and 18 ml ClNaO 4%), then pelleted for 1 minute at 300 g. This bleaching procedure was repeated two more times, corresponding to a total of 8-12 minutes in bleaching solution, in order to lyse the adult worms and release embryos (about 0.6-0.8 g). After bleaching, the embryos were washed twice with M9 medium.
The remaining steps were performed at 4°C and are based on our previously described methods for isolating protein complexes from yeast cells 1 (link), 10 (link). Embryos were washed twice with lysis buffer (100 mM HEPES-KOH pH 7.9, 50 mM potassium acetate, 10 mM magnesium acetate, 2 mM EDTA), and then resuspended with three volumes of lysis buffer that was supplemented with 2 mM sodium fluoride, 2 mM sodium β-glycerophosphate pentahydrate, 1 mM dithiothreitol (DTT), 1% Protease Inhibitor Cocktail (P8215, Sigma-Aldrich), and 1X ‘Complete Protease Inhibitor Cocktail’ (05056489001, Roche; one tablet dissolved in 1 ml water makes a 25× stock solution). The mixture was transferred drop-wise into liquid nitrogen to prepare ‘popcorn’, which was stored at -80°C. We then ground ~2.5 g of the frozen popcorn in a SPEX SamplePrep 6780 Freezer/Mill. After thawing, we added one-quarter volume of ‘glycerol mix’ buffer (lysis buffer supplemented with 50% glycerol, 300 mM potassium acetate, 0.5% detergent IGEPAL CA-630, protease inhibitors, and DTT at the concentrations mentioned above). De-ubiquitylase enzymes were inhibited by addition of 5µM Ubiquitin PrG (prepared by Axel Knebel and Clare Johnson, MRC PPU, Dundee), and chromosomal DNA was digested with 1600 U of Pierce Universal Nuclease (123991963, Fisher) for 30 minutes at 4°C. Extracts were centrifuged at 25000 g for 30 minutes and then for 100000 g for 1 hour, before pre-incubation with agarose beads (0.4 ml slurry) for 45 minutes. At this point, 50 µl of extract was added to 100 µl of 1.5X Laemmli buffer and stored at -80°C. The remainder of the extracts were then incubated for 90 minutes with 40 µl of GFP-Trap_A beads (Chromotek). The beads were washed four times with 1 ml of wash buffer (100 mM HEPES-KOH pH 7.9, 100 mM potassium acetate, 10 mM magnesium acetate, 2 mM EDTA, 0.1% IGEPAL CA-630, 2 mM sodium fluoride, 2 mM sodium β-glycerophosphate pentahydrate, plus protease inhibitors as above) and bound proteins were eluted at 95°C for 5 min in 100 µl of 1x Laemmli buffer (or 50 µl when used for mass spectrometry analysis) and stored at -80°C.
For each experiment, 1ml of a synchronized population of L4 worms expressing GFP-PSF-1 were fed for 50 hours at 20°C on a 15 cm RNAi plate (see above), supplemented with 8 g of bacterial pellet for the required RNAi treatment, prepared as described above. After feeding, the adults worms were washed in M9 medium and resuspended for 2 minutes at room temperature in 14 ml of ‘bleaching solution’ (for 100 ml: 36.5 ml H2O, 45.5 ml 2N NaOH and 18 ml ClNaO 4%), then pelleted for 1 minute at 300 g. This bleaching procedure was repeated two more times, corresponding to a total of 8-12 minutes in bleaching solution, in order to lyse the adult worms and release embryos (about 0.6-0.8 g). After bleaching, the embryos were washed twice with M9 medium.
The remaining steps were performed at 4°C and are based on our previously described methods for isolating protein complexes from yeast cells 1 (link), 10 (link). Embryos were washed twice with lysis buffer (100 mM HEPES-KOH pH 7.9, 50 mM potassium acetate, 10 mM magnesium acetate, 2 mM EDTA), and then resuspended with three volumes of lysis buffer that was supplemented with 2 mM sodium fluoride, 2 mM sodium β-glycerophosphate pentahydrate, 1 mM dithiothreitol (DTT), 1% Protease Inhibitor Cocktail (P8215, Sigma-Aldrich), and 1X ‘Complete Protease Inhibitor Cocktail’ (05056489001, Roche; one tablet dissolved in 1 ml water makes a 25× stock solution). The mixture was transferred drop-wise into liquid nitrogen to prepare ‘popcorn’, which was stored at -80°C. We then ground ~2.5 g of the frozen popcorn in a SPEX SamplePrep 6780 Freezer/Mill. After thawing, we added one-quarter volume of ‘glycerol mix’ buffer (lysis buffer supplemented with 50% glycerol, 300 mM potassium acetate, 0.5% detergent IGEPAL CA-630, protease inhibitors, and DTT at the concentrations mentioned above). De-ubiquitylase enzymes were inhibited by addition of 5µM Ubiquitin PrG (prepared by Axel Knebel and Clare Johnson, MRC PPU, Dundee), and chromosomal DNA was digested with 1600 U of Pierce Universal Nuclease (123991963, Fisher) for 30 minutes at 4°C. Extracts were centrifuged at 25000 g for 30 minutes and then for 100000 g for 1 hour, before pre-incubation with agarose beads (0.4 ml slurry) for 45 minutes. At this point, 50 µl of extract was added to 100 µl of 1.5X Laemmli buffer and stored at -80°C. The remainder of the extracts were then incubated for 90 minutes with 40 µl of GFP-Trap_A beads (Chromotek). The beads were washed four times with 1 ml of wash buffer (100 mM HEPES-KOH pH 7.9, 100 mM potassium acetate, 10 mM magnesium acetate, 2 mM EDTA, 0.1% IGEPAL CA-630, 2 mM sodium fluoride, 2 mM sodium β-glycerophosphate pentahydrate, plus protease inhibitors as above) and bound proteins were eluted at 95°C for 5 min in 100 µl of 1x Laemmli buffer (or 50 µl when used for mass spectrometry analysis) and stored at -80°C.
