Dye plus

Arabidopsis plants were transformed with the floral dip method (Clough and Bent, 1998 (link)) using A. tumefaciens GV3101 carrying the 35S::ZmNBS25 construct or the control vector. Transformed Arabidopsis seeds were sown onto Murashige and Skoog (MS; Sigma-Aldrich) agar plates containing 20 mg mL⁻¹ hygromycin (Roche Molecular Diagnostics) to screen for positive transformants, which were then transplanted for further growth. Genomic DNA was extracted from the leaves of transgenic Arabidopsis lines using the CTAB (cetyl trimethyl ammonium bromide) method (Clarke, 2009 (link)). The DNA extracted from each transgenic Arabidopsis plant was used as template to determine positive transgene integration by PCR. The 2× Taq Master Mix (Dye Plus; Vazyme Biotech Co., Ltd., Nanjing City, PRC) was used for PCR amplification under the following profile: 95°C for 10 s, 32 cycles of 55°C for 30 s, and 72°C for 30 s. Seeds from positive transformants were harvested, subjected to hygromycin screening, and the process was repeated until only seeds capable of growing in hygromycin medium remained. The homozygous T3 generation was selected for subsequent experiments.

RT PCR for AKT3 transcripts was performed as previously reported [15 (link)]. Total RNA (2 μg) extracted from the hippocampi of aged mice was reverse transcribed to cDNA using the Hifair® III 1st strand cDNA synthesis kit containing gDNA digester plus (Yeasen, China). The hippocampi of mice were injected with the recombinant adeno-associated virus to overexpress circAKT3 (rAAV-hSyn-mmu-circAKT3-nEF1α-EGFP) or the empty vector (rAAV-hSyn-EGFP) to exclude the interference of rAAV injections. Next, cDNA was amplified using 2 × Phanta^® Max Master Mix with Dye Plus (Vazyme, China) and common sense primer (5′-GGACTATCTACATTCCGGAAAG-3′) with AKT3 transcript 1 primer (5′-GGTGAAGACCCTTGGCTGGTC-3′) or AKT3 transcript 2 primer (5′-GGGTCTAGATTACTTTTTATTATCATTTTTTTTCCAGTTAC-3′). The primer sequences were synthesized as reported previously [15 (link)]. And the PCR protocol consisted of preliminary denaturation (95 ℃, 3 min), repeated 35 cycles containing denaturation (95 ℃, 15 s), annealing (54 ℃, 15 s) and extension (72 ℃, 30 s), and a further extension at 72 ℃ for 5 min. Afterwards, the Semiquantitative RT-PCR was scored by agarose gel electrophoresis (1%) mixed with GelRed nucleic acid dye (Yeasen, China) and photographed by the ultraviolet imaging system.

Genomic DNA was harvested using a Mouse Director PCR Kit (Bimake). PCR amplification of KMT2D-LCDs was performed using 2 × Taq Master Mix (Dye Plus; Vazyme). PDLIM7 was used as the positive control to determine the concentration of high-quality DNA isolated from the modified cells. All primers in this study are listed in Additional file 1: Table S1.