GSM8140792: WT_N2_Day2_biol_rep3; Caenorhabditis elegans; ncRNA-Seq
Synchronous populations of worms were grown at 20°C on NGM plates seeded with OP50 E. coli concentrated food at a density of maximum 40,000 animals per 15 cm Petri dish and harvested at Young Adult stage at 48 hours post hatching. The harvested animals were washed three times with M9 buffer and 40 µl of worm pellet was frozen in dry ice with TRI Reagent (MRC, Inc.). After five repetitions of freeze and thaw, total RNA was isolated according to the TRI Reagent protocol. For the 2-cell embryo RNA extraction, precisely staged 2-cell embryos were obtained by alkaline hypochlorite treatment of synchronized gravid adult hermaphrodites, and fifty 2-cell embryos were collected using a mouth pipet. Embryos were lysed for 10min at 65˚C and 1min at 85˚C in a lysis solution containing Tris pH8 5mM final, EDTA 0.25mM final, Triton 100X 0.5%, Tween 20 0.5%, Proteinase K 0.5% and Water. The lysates were subjected to RNA purification using the Quick-RNA MicroPrep kit (ZymoResearch, R1051), and small RNAs (RNA fraction from 17 to 200 nucleotides) and mRNAs (RNA fraction >200 nucleotides) were collected separately. A small RNA spike-in was added before embryo lysis. The small RNA library preparation was performed essentially as described previously by Barucci et al., 2020.