Quantification of pfeRNA Expression Levels

The whole process of the evaluation of pfeRNA expression levels was similar to what we described before [17 (link),18 (link),20 (link)], and the process included adaptor ligation, RT, and QuantStudio Dx PCR. The adaptor/5′rapp/5′-CTGTAGGCACCATCAAT-3′/3′ddc/with both 5′ and 3′ modification, meaning only the 3′-end of sncRNA was ligated. Specifically, 5 µL of total sncRNAs and 1 µL (2 µM) of adaptor were ligated using single-strand truncated T4 RNA ligase 2 (New England Biolabs, cat# M0242L) overnight at 16 °C, and the ligation reaction was terminated at 65 °C for 15 min. For RT, the SuperScript II First-Strand Synthesis System (ThermoFisher Scientific, cat# 18064) was used according to the manufacturer’s instructions with gene-specific reverse transcription primer, and the total volume was 20 µL after RT. For QuantStudio PCR, a common reverse primer and primers specific for individual pfeRNA were used, and the amplification quality of each pair of primers was determined by both generating the melting curves and amplification curves. Each sample was tested in triplicate, and the total volume of each reaction was 20 µL. Amplification conditions were denaturation at 95 °C for 15 s (15 min for the first cycle), annealing at 60 °C for 20 s, extension at 72 °C for 20 s, and 40 cycles. All primers and adaptor are listed in Supplementary Table S1.