5prime phase lock gel

Total RNA was extracted from NK-92 cells with phenol/chloroform, followed by cleaning step with 5Prime Phase Lock Gel (Quantabio, Beverly, MA, US) and ethanol precipitation. One microgram of total RNA was reverse transcribed using oligo(dT)₁₈ primers with RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Two-step qPCR reactions were performed in triplicates using Maxima SYBR Green/ROX qPCR Master Mix 2X (Thermo Fisher Scientific, Waltham, MA, USA) by adding 1.0 ng cDNA at 10 µL volume and were run with LightCycler 480 (Roche Diagnostics, Basel, Switzerland) at following conditions: 10 min at 95 °C, followed by 40 cycles at 95 °C for 15 sec and at 60 °C for 1 min. Primers were designed with NCBI Primer-BLAST (accessed on 19 February 2020)-Table 1, validated by melting curve analysis and reaction efficiencies were confirmed to be within 90% to 110%. GAPDH was selected as the optimal reference gene by NormFinder [59 (link)]. Data were calculated according to the guidelines by Taylor et al. [60 (link)].

Cells were transfected at 60 to 80% confluence with FuGENE HD^®according to the manufacturer’s protocol at a 3:1 FuGENE HD^®to DNA ratio. For high-throughput measurements of splicing outcomes, 10 $\mathrm{\mu }$ g pooled reporter assay DNA was transfected in three 100-mm plates. For biochemical analysis of individual reporters, 1 $\mathrm{\mu }$ g or 2.5 $\mathrm{\mu }$ g individual reporter DNA was transfected into each well of a 12- or 6-well plate (respectively). Then, 24 h after transfection, total RNA was isolated from detached cells (Accutase^®, ThermoFisher). For amplicon sequencing, total RNA was isolated using phenol-chloroform (Ambion) extraction (5PRIME Phase Lock Gel, Quantabio) followed by DNase treatment (TURBO DNase). For biochemical analysis, RNA was isolated using a silica column (illustra™RNAspin Mini RNA Isolation Kit, GE Healthcare) with on-column DNase digestion following the manufacturer’s automated protocol. DNase-treated RNA was reverse transcribed using a reporter-specific primer following the manufacturer’s specifications (SuperScript IV Reverse Transcriptase, Thermo Fisher) with RNase H treatment. Reverse transcription primers included degenerate nucleotides to serve as unique molecular identifiers (UMIs) during amplicon sequencing (46 (link), 47 ). cDNA products were used for amplicon sequencing or biochemical analysis.