GSM8140817: L1_DP_AID_EtOH_biol_rep2; Caenorhabditis elegans; RNA-Seq
To obtain synchronized non-starved L1, we used the NemaSync C. elegans synchronizer model 5000. L1 larvae were frozen in dry ice with TRIzol Reagent (Invitrogen), and after six repetitions of freeze and thaw, RNA was extracted using the Direct-zol RNA Microprep kit (ZymoResearch, R2062). The small RNA fragments (17 to 200 nucleotides) were separated from the large RNAs (>200 nucleotides) using the Quick-RNA MicroPrep kit (ZymoResearch, R1051). The large RNA was DNAse treated using the reagent provided along with the Quick-RNA Microprep kit. Using these size separation kits, we were able to sequence both the small RNA and the mRNA coming from the same samples. An Agilent 2200 TapeStation System was used to evaluate the RIN indexes of the large RNAs (>200 nucleotides), and only samples with RNA integrity number RIN  > 8 were used for RNA-seq. For 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. The RNA fraction > 200 nucleotides were used for preparing RNA-seq libraries. DNase-treated total RNA with RIN > 8 was used to prepare strand-specific RNA libraries. Ribosomal and mitochondrial rRNAs were depleted using a custom RNAse-H-based method to degrade rRNAs using complementary oligos as described in42.Strand-specific RNA libraries were prepared using at least 100 ng of rRNA-depleted RNAs using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760S). RNA libraries were analysed on Agilent 2200 TapeStation System using high sensitivity D1000 screentapes and quantified using the Qubit Fluorometer High Sensitivity dsDNA assay kit (Thermo Fisher Scientific, Q32851). Multiplexed libraries were sequenced on a NextSeq 2000 Illumina platform.