Worm Harvest:

For the germ line experiments, 5-8 150mm plates were harvested for each timepoint, and provided sufficient RNA for at least one, and usually many more, hybridizations. To these plates, 8 ml M9 (6g Na2HPO4, 3g KH2PO4, 5g NaCl, 0.25g MgSO4 per liter) was added to each plate. To avoid temperature shock, the M9 was pre-incubated at 25°C. The plates were slowly rotated on a shaker for 2 minutes, and then the M9 and worms were pipetted into 15ml Falcon tubes and spun at the fastest setting in a clinical centrifuge for ~1 minute. During this spin, the plates were washed a second time in 5ml of M9 per plate to collect the residual worms. After removal of the supernatant from Falcon tubes, the second wash was added and the tubes spun again. The number of tubes was consolidated by washing the pellets with fresh M9 once or twice more, and combining the pellets after each wash. The final pellet size in a tube was not more than 2.5ml in a 15ml conical. At this point, the first few steps of the RNA isolation were completed.