title: GSM8634265: Ubiquitous depletion of RPC-1, Day 1 adult hermaphrodites, NGM + 4mM auxin, rep 3; Caenorhabditis elegans; ncRNA-Seq
library_construction_protocol: Worm pellets of Day 1 adults were snap frozen and stored in -80C until samples were ready to be prepared. At the time of RNA isolation, worm pellets were resuspended in Trizol, followed by three freeze-thaw cycles on dry ice, and vortexing for 15 minutes at room temperature. Thereafter, total RNA was purified according to the Trizol manufacturer's instructions (ThermoFisher). Spike-ins were added at a final concentration of 0.1 pg/μl in 100 ng/μl total RNA. sRNA libraries were constructed using NEBNext Small RNA Library Prep Set for Illumina with modifications. After reverse transcription, each sample was treated with 5000units of RNAse H (New England Biolabs) for 20 minutes at 37C. The treated samples were immediately run on 8% urea gels to purify and size select ~65-75nt RT products.