The role of MLF1 on accessible chromatin in AC16 cell line

Previous studies found that MLF1 mRNA levels tended to decline in aging hearts. Functional studies found that knockdown of MLF1 ameliorated the drug-induced cellular senescence phenotype. Here, we apply transcriptomics to explore the underlying molecular mechanisms. Cardiomyocyte AC16 cells were cultured in DMEM/F12 . DMEM/F12 supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 100 U/mL penicillin, and 100 μg/ml streptomycin. Medium without FBS was replaced before transfection. Negative control siRNA and Mlf1 siRNA were transfected into AC16 cells at ~70% confluence using jetPRIME® (Polyplus-transfection, NY, USA). After 12 h of transfection, fresh complete medium was replaced. Accessible chromatin mapping was performed using the ATAC-seq method as previously described. About 50000 AC16 cells were lysed in ATAC-seq lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.5% NP-40) and centrifuged for 10 min at 4°C. Then, nuclei were spun at 1500g for 5 min to remove the supernatant. Nuclei were then incubated with the Tn5 transposome and tagmentation buffer at 37 °C for 30 min using TruePrep® DNA Library Prep Kit V2 for Illumina (Vazyme, TD501-01). After DNA purification with the MinElute kit, and the PCR was performed to amplify the library for 12 cycles and libraries were purified with the MinElute kit. Amplified ATAC libraries were sequenced on the Illumina Novaseq 6000 platform.