GSM5765424: lotr1_sam117_r1; Caenorhabditis elegans; miRNA-Seq
For RNA extraction, 500 µl of Trizol LS (Life Technologies) was added to 50 µl of worms and frozen for 30 s in liquid nitrogen followed by thawing for 2 min at 37°C. Freeze-thaw cycles were repeated until worms were dissolved. Samples were centrifuged for 5 min at 14800g and supernatants were mixed 1:1 with cold 100% ethanol. RNA isolation was performed with Zymo Research Direct-zol RNA microprep kit (R2062) according to manufacturer's protocol and 1 µg RNA of each sample was subsequently treated with RNA 5´ Pyrophosphohydrolase (RppH, NEB) as described in Almeida et al. 2019 (https://doi.org/10.1016/j.mex.2019.01.009). NGS library prep was performed using the NEXTflex Small RNA-Seq Kit V3 following Step A to Step G of Bioo Scientific`s standard protocol (V19.01) with a ligation of the 3' 4N Adenylated Adapter overnight at 20°C. Libraries were prepared with a starting amount of 580 ng RppH-treated RNA per sample, amplified in 16 PCR cycles, purified on an 8% TBE gel and size-selected in the range 144-163 bp. Purified libraries were profiled with a High Sensitivity DNA kit on a 2100 Bioanalyzer (Agilent), quantified using the Qubit dsDNA HS Assay Kit in a Qubit 2.0 Fluorometer (Life Technologies), pooled in equimolar ratio and sequenced on a NextSeq 500/550 Illumina sequencer.