Small RNA-seq of Caenorhabditis elegans young adults: slfl-3 deletion, CIP and RppH treated, experiment 3, replicate 3
L1 worms were seeded on NGM OP50 plates and grown at 20 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 (Termo Fisher) 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-zol RNA Microprep Kit (Zymo Research) according to its protocol. For the analysis of piRNA precursors, CIP treatment on 1,5 ug of isolated RNA was performed in rCutSmart Buffer (NEB) using 3 uL of Quick CIP (NEB) in a 40 uL reaction. The reaction was incubated at 37 C for 20 min followed by heat inactivation for 2 min at 80 C. The CIP-treated RNA was subjected to another round of purification using the Direct-zol RNA MicroPrep kit (Zymo Research). RppH (NEB) treatment was performed with a starting amount of 500 ng. 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). Libraries were prepared with a starting amount of 200 ng and amplified in 18 PCR cycles. Amplified libraries were purified by running an 8% TBE gel and size-selected for 15-40 bp insert length. Libraries were profiled in a High Sensitivity DNA Chip on a 2100 Bioanalyzer (Agilent Technologies), quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life Technologies), and sequenced on Illumina NextSeq 500 sequencer.