Generation of KMT2D Knockout H9c2 Cells

The generation of KMT2D knockout H9c2 cardiomyocytes cell lines based on CRISPR/Cas9 gene-editing knockout system has been reported in our previous study [14 (link)]. Briefly, the oligodeoxynucleotides of gRNAs were annealed and cloned into PX458 plasmids (48138, Addgene), named PX458-Kmt2d-gRNA1 and PX458-Kmt2d-gRNA2, respectively, and confirmed by sequencing. H9c2 cells were detached by 0.25% trypsin–EDTA solution (25200–056, Gibco) and transfected with CRISPR/Cas9 PX458-Kmt2d gRNA1/2 or empty control plasmids by Nucleofector™ X Kit (V4XC-2024, Lonza) according to the manufacture’s protocol, then plated in DMEM with 10% FBS without pen/strep. Cells were cultured for 48 h at 37 °C. The PX458 plasmid contained a green fluorescent protein (GFP) marker that can be used to screen GFP-positive cells for Kmt2d-KO monoclonal H9c2 cell line by aseptic flow cytometry.