Transfection and RT-PCR analysis
The minigene plasmid transfected into HEK293T cells with lipofectamine 3000 reagent (Thermo Fisher Scientific, USA) based on manufacturer’s instruction. After 48h, collected the cells and then performed RT-PCR to evaluate the mRNA splicing. Briefly, total RNA was extracted using TRIzol reagent (Sigma-Aldrich) and then reverse transcribed to cDNA by reverse transcription kit (Vazyme) according to the instruction. PCR was performed to amplify the fragment containing the mutant site. All primers were listed below: forward: 5’- ATGGTGGTGTTCGGGTATGA-3’, reverse: 5’-AGCGTCTGAACAG GAAGTTTG-3’ (for lymphocytes, exon 1-3); forward: 5’-GAAGCGCCCAAGGTTTTCAA-3’, reverse: 5’-ACTATGCCAATCAGTGAGCTTCT-3’ (for lymphocytes, exon 2-4); dSD-F: 5’-TCTGAGTC ACCTGGACAACC-3’, dSA-R: 5’-ATCTCAGTGGTATTTGTGAGC-3’ (for HEK293T cells). Agarose gel electrophoresis (BioFroxx, Germany) was used to separate the segment of PCR products. Then the sequence of each DNA product was determined by sanger sequence.