RT-qPCR validation of differential alternative splicing events

The differential alternative splicing of MMD and ADA was validated by duplex RT-qPCR using customized primers and probes. Primers and TaqMan^® probes (Life Technologies, Carlsbad, CA) were designed based on each event using Custom TaqMan^® Assay Design Tool (https://www.thermofisher.com/). The primers and probes are presented as followed (Supplementary Table S5):

Isoform 1 of MMD: Forward primer: 5′-AATGGCCGCTACAAGCCAAC-3′; Reverse primer: 5′- CATCAGAGAGCCGGTGAAGG-3′; Probe: 5′FAM-AATGGCCGGAACAATGAGGAATGC-NFQ-MGB3′;

Isoform 2 of MMD: Forward primer: 5′-CAGTGCTGATCCTATCTGGAAGA-3′; Reverse primer: 5′-CATCAGAGAGCCGGTGAAGG-3′; Probe: 5′VIC-AGTTTGCATTCTTTCCTCATTGTTCCGG-NFQ-MGB3′;

Isoform 1 of ADA: Forward primer: 5′-TGTGGAGATGAAGGCCAAGG-3′; Reverse primer: 5′-CCAGTGACACCACCTCATCC-3′; Probe: 5′FAM-AGCCGATCCCCTGG AACCAGGCTGAAGGGG-NFQ-MGB3′;

Isoform 2 of ADA: Forward primer: 5′-CTCCTTCCTCTCTCTCCTACC-3′; Reverse primer: 5′-GATGAGGTGGTGTCACTGG; Probe: 5′VIC-TTCCCCACACACAGAGGGGACCTCACCCCG-NFQ-MGB3′.

Total RNA (1.0 μ g) from each sample was treated with DNAase I (Invitrogen), and reverse-transcribed to cDNA was performed using SuperScript II reverse transcriptase following the manufacturer’s protocol (Invitrogen). A total of 100 ng cDNA, 10.0 ptase fol^® Fast Advanced Master Mix, primers (final concentrations was 900 nM) and probe (final concentration was 250 nM) were used in a 20 μl reaction, which was suggested by TaqMan^® Gene Expression Assays Protocol. Primers and probes for the two isoforms of MMD or ADA were added to one reaction. The fluorescence signals were detected with StepOnePlus Real-Time PCR System (Life Technologies) with the following cycling times and temperatures: 50 °C for 2 mins, 95 °C for 20 s and 40 cycles of 94 °C for 1 s, 60 °C for 20 s. The cycle threshold (Ct) values for the isoforms in different samples have been listed in Supplementary Table S6. The alternative splicing events were detected by evaluating the ratio between the expressions of the two isoforms: Expression _{isoform 1}/Expression _{isoform 2} = 2^{(Ct isoform 2 -Ct Expression even)}.