GSM5241337: N2_adult_r2; Caenorhabditis elegans; miRNA-Seq
For RNA extraction, worms were thawed and M9 was replaced with 250 µL of worm lysis buffer (0.2M NaCl, 0.1M Tris pH 8.5, 50 mM EDTA, 0.5% SDS), supplemented with 300 µg Proteinase K (Sigma-Aldrich, P2308). Subsequently, lysis was conducted for approximately 90 minutes at 65oC. Next, 3 volumes of TRIzol LS (Life Technologies, 10296-028) were added and subsequent isolation was as defined by the manufacturer's instructions. Samples were enriched for small RNAs using a mirVana Kit (Life Technologies, AM1561). Samples were subsequently treated with RppH as described in Almeida et al. 2019 (https://doi.org/10.1371/journal.pgen.1007784).  Then, 1 µg of RppH-treated RNA was loaded on a 15% TBE-urea gel and size-selected between 16- to 30-nt. Purified fraction was confirmed by Bioanalyzer sRNA chip (Agilent). Small RNA library preparation was based on the NEBNext Multiplex sRNA Library Prep Set for Illumina (New England BioLabs) with slight modifications. To counteract ligation biases, the 3' and 5' adapters contained 4 random bases at the 5'- and 3'-end, respectively, and were chemically synthesized by BioScientific. Adapter-ligated RNA was reverse-transcribed and PCR-amplified for 14 cycles using index primers. The PCR-amplified cDNA construct was purified using AMPure XP beads (Beckman Coulter). The purified PCR reaction was checked on the Bioanalyzer using High Sensitivity DNA chip (Agilent). Size selection of the sRNA library was done on LabChip XT instrument (Perkin Elmer) using DNA 300 assay kit. Only the fraction containing 140-165 bp was pooled in equal molar ratio. The resulting 10 nM pool was denatured to 10 pmol with 5% PhiX spike-in and sequenced as single-read on HiSeq 2500 (Illumina) in rapid mode for 51 cycles (plus 7 cycles index read)  using on-board cluster generation.