GSM9116798: control rep. 2; Caenorhabditis elegans; RNA-Seq
60,000 L1 larvae were plated to 150 mm NGM plates supplemented with 2 mL of concentrated OP50 food. Approximately 64 hours after plating L1 larvae, day 1 adults with eggs were collected from plates using M9 solution. Harvested animals were washed twice with M9 and once with water. Animals were then suspended in TRI Reagent solution (Sigma) and frozen at -80°C until RNA extraction was performed. To extract RNAs animals suspended in TRI Reagent solution were thawed and lysed using a Bead Mill (Thermo Fisher Scientific). Bromo-chloropropane (Sigma) was added to the lysed worms and centrifuged at 12,000 x g for 15 minutes. Following centrifugation, the aqueous phase was pipetted to an equal volume of cold isopropanol and placed at -20°C for 1 hour to precipitate RNAs. RNAs were pelleted by centrifugation at 20,000 x g for 15 minutes at 4°C. Total RNAs were washed with 75% EtOH twice and suspended in RNAse free water. Ribosomal RNAs (rRNAs) were depleted using the RNase H-based method. Specifically, 3 μg of total RNA was incubated with antisense DNA oligonucleotides targeting C. elegans and E. coli rRNAs, followed by digestion with Thermostable RNase H (NEB) at 45 °C for 30 min. The rRNA-depleted samples were treated with Turbo DNase (Thermo Fisher Scientific) at 37 °C for 30 min. RNAs longer than 200 nts were enriched using RNA Clean & Concentrator-5 (Zymo Research). Strand-specific mRNA sequencing libraries were constructed using Ultra II Directional RNA Library Prep Kit (NEB), according to the manufacturer's instructions. Each library was barcoded, pooled, and sequenced on the HiSeq 4000 platform (2x150 bp, Illumina).