Mission shrna custom lentiviral particles

Short-hairpin(sh) approach using lentiviral particles was used for studies to achieve the silencing of genes of interest. Silencing DIAPH1 and AGER in HiPSC-CMs and DIAPH1 in H9C2 and HMVECs was achieved using mission shRNA custom lentiviral particles from Millipore Sigma (Supplementary Table 8). Short-hairpin targeting scrambled (Scr) sequence was used as a control for studies. For all cell types, transduction was performed at a confluency of 30–40%. In total, 1.5 × 10⁶ viral particles of either Scr or gene of interest at an approximate multiplicity of infection (MOI) of 3 were used for transduction. Hexadimethrine bromide (Sigma cat# 107689) at a concentration of 8 µg/ml was used to increase lentiviral uptake and transduction efficiency. Cells were incubated for 5–6 h with Lentiviral particles to obtain excellent transduction efficiency, and thereafter, cultures were replaced with regular growth medium. Forty-eight hours post transduction, cells were selected against puromycin resistance (Sigma cat# P8833) at a concentration of 2 ng/ml for successful transduction. In the case of HiPSC-CMs, due to poor transduction efficiency, precursor HiPSCs were initially transduced and tested for silencing efficiency by qPCR. Upon achieving successful transduction, the cells were differentiated into HiPSC-CMs for experiments.