2 step cells to ct

HEK293T cells were plated at 20,000 cells per well in 96-well culture plates (VWR). Each well was transfected at 75% confluency with 250 ng TRM editor plasmid and 150 ng Cas13 guide RNA plasmid using 0.5 μL Lipofectamine 2000 (Thermo Fisher) according to the manufacturer’s protocol. As a vector control, 400 ng of pUC19 plasmid was also transfected. After 48 h post-transfection, RNA was isolated and converted to cDNA using a 2-step Cells-to-Ct (Thermo Fisher) kit with 10 min (2× longer than suggested) DNaseI treatment to ensure degradation of genomic DNA. All other steps were carried out according to manufacturer’s protocol. Semi-quantitative PCR was performed in 20 μL reactions with Q5 Hot-Start High-Fidelity DNA Polymerase (New England BioLabs), 1 μL of cDNA, and 0.5 μM of each forward and reverse primer (Integrated DNA Technologies, Supplementary Table 6). All PCR reactions were performed for 35 cycles. Amplified cDNA was diluted 1:20 in nuclease-free H₂O then run on a Tapestation D1000 or D5000 screen tape (Agilent Technologies). Size abundances of each splicing isoform were integrated to quantify exon exclusion and inclusion.