Protoscript first strand cdna synthesis kit

DNase-treated RNA samples (1µg) were reverse transcribed to complementary DNA (cDNA) using the Protoscript^® First Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA) and following the manufacturer’s protocol. cDNA samples (1µg) from the patient and non-affected father (control sample), as well as the respective RNA samples not reverse transcribed (RT-ve) and a no-template (water) sample, were PCR amplified using HotStarTaq Plus DNA polymerase (QIAGEN) and custom reverse-transcription PCR (RT-PCR) primers (Metabion) (Table S4). The integrity of the cDNA samples was tested by amplification of a 215 bp fragment from the β-actin gene (ACTB) (Table S4). All RT-PCR products were loaded on a 2% agarose gel, which was pre-stained with GelRed^® (1× final concentration) (Biotium, Fremont, CA, USA) and run at 120V for ~1 h. Selected RT-PCR products of interest were purified and further processed with bidirectional Sanger sequencing as described above. The specific RT-PCR reaction volumes and conditions used are available upon request.

Quantitative real-time reverse transcription-PCR was performed according to manufacturer's instructions (Lightcycle 96, Roch, Germany)^{19 (link)}. In brief, total RNA was isolated from cells using RNeasy kit from Qiagen. cDNA was generated by reverse-transcription of equivalent quantities of RNA using ProtoScript First Strand cDNA synthesis kit from NEB. qRT-PCR was performed using SYBR Green PCR master mix (qPCRBIO SyGreen Mix Lo-Rox, Nippon Genetics, Germany). Primer sequences are listed in Supplementary Table 1. Actin was used as an endogenous control.

RNA was isolated by lysing worms with protK followed by TRIzol LS extraction. In RIP experiments, TRIzol LS was added directly to the immunoprecipitation beads after washing. Quantitative PCR was done in an Applied Biosystems ViiA7 real-time PCR system (Thermo Fisher Scientific) with cDNA made from 500 ng of total RNA using ProtoScript first strand cDNA synthesis kit (New England Biolabs, E6300) and Oligo d(T)₂₃VN. Next-generation sequencing library preparation was performed with NEXTflex small RNA-seq kit version 3 following step A to step G of Bioo Scientific`s standard protocol (version 16.06). Further details on these protocols, including details on bioinformatic analysis of next-generation sequencing data, are in the Supplemental Material.