title: Small RNA-seq of Caenorhabditis elegans young adults: PID-3 point mutant I182AV186A, grown at 25 degree, replicate 3
design: L1 worms were seeded on NGM OP50 plates and grown either at 20 or 25 C until young adult stage, washed with M9 buffer and snap frozen in liquid nitrogen in 50 ul aliquots. For RNA purification, worm aliquots were thawed on ice, 500 ul of TRIzol LS Reagent was added and samples underwent five freezing-thawing cycles in liquid nitrogen followed by centrifugation. Supernatants were collected and RNA purification was carried out with Direct-zo RNA Microprep Kit according to its protocol. NGS library prep was performed with NEXTflex Small RNA-Seq Kit V3 following Step A to Step G of Bioo Scientific`s standard protocol (V19.01) with a starting amount of 30 ng, amplified in 21 PCR cycles, purified by running on a 8% TBE gel and size-selected in the range 144  163 nt. The libraries were profiled using a High Sensitivity DNA kit on a 2100 Bioanalyzer (Agilent Technologies) and quantified using the dsDNA HS Assay Kit in a Qubit 2.0 Fluorometer (Life Technologies). All samples were pooled in equimolar ratio and sequenced on a NextSeq 500/550 Flowcell, SR for 75 cycles plus 7 cycles for the index read.