Single strand truncated t4 rna ligase 2

The whole process of evaluating pfeRNA expression levels was similar to that we described before^{4 (link),7 (link),17 (link),25 (link)}. Specifically, the method includes adaptor ligation, RT, and QuantStudio PCR. An adaptor with both 5′ and 3′ modifications ligates only the 3′-end of sncRNA and enhances the ligation efficiency. For each ligation reaction, 5 µl total sncRNAs and 1 µl (2 µM) adaptor were ligated using single-strand truncated T4 RNA ligase 2 (New England Biolabs, cat# M0242 L) 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 (Thermo Fisher Scientific, cat# 18064) and gene-specific reverse primers were used, and the total volume was 20 µl after RT. For QuantStudio PCR, a common reverse primer and primers specific for individual pfeRNAs were used. Each sample was tested in duplicate, 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 using a QuantStudio 3 machine (Applied Biosystem in Thermo Fisher Scientific). Each experiment was repeated three independent times. All primers and adaptors are listed in Supplementary Table S2.

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.