Total rnapure kit

Total RNA was extracted from fresh leaf samples using the Total RNApure Kit (ZOMANBIO, Beijing, China). The first-stand cDNA was synthesized using TRUE RT Master Mix (AidLab Biotech, Beijing, China) following the manufacturer’s instructions. PCR was conducted using appropriate primers (Supplementary data Table S3) and Phanta Max Master Mix (Vazyme, Nanjing, China) on a C1000 Thermal Cycler (Bio-rad Laboratories, Hercules, USA), while quantitative PCR was performed using Bio-Rad CFX96™ Real-time System (Bio-rad Laboratories) with 2× qPCR Real-Time Kit (Applied Biological Materials (abm) Inc., Vancouver, Canada). The 2^-ΔΔCq method was used for gene expression level analysis based on potato internal reference gene elongation factor 1 alpha (EF1α, accession number: Soltu.DM.06G005620) in RT-qPCR [83 (link)]. All histograms were made using GraphPad Prism.

RNA isolation from the frozen samples was performed using a Total RNApure Kit (ZOMANBIO, http://zomanbio.com). Reverse transcription into cDNA was carried out using 5× All-in-One RT MasterMix (ABM, http://www.abmgood.com). Quantitative RT-PCR was performed with the Bio-Rad CFX Connect^TM Real-Time System (Bio-Rad, http://www.bio-rad.com) and EvaGreen 2X qPCR MasterMix (ABM, http://www.abmgood.com). The potato ef1α gene (GenBank accession: AB061263) was used as a control gene for normalization of expression^{38 (link)}. Gene expression levels were calculated via the 2^−Δcq method described by Bio-Rad (http://www.bio-rad.com/zh-cn/applications-technologies/real-time-pcr-experimental-design). All primer sequences for qRT-PCR analysis are detailed in Supplementary Table S1.

RNA m⁶A dot blotting was employed to verify m⁶A binding with anti-m⁶A antibody. HT22 cells were collected at 95% confluence, and total RNA was isolated and purified according to the standard protocol of the Total RNApure Kit (#ZP404-1, ZOMANBIO, China). The 6 μg RNA suspended in 200 μL RIP lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40) supplemented with 0.2 μL RNase inhibitor (#N8080119, Thermo Fisher Scientific), and incubated overnight at 4 °C with magnetic beads bound with 5 μg of rabbit anti-m⁶A (#202003, Synaptic Systems, Germany) or rabbit anti-IgG (#AC005, ABclonal, China) antibodies. After IP, the aliquots of sample solution were then detected by dot blotting analysis to verify m⁶A binding. Briefly, the aliquots above were added to the nitrocellulose membrane (#66485; Biosharp, China). The membrane was then cross-linked by UV light, sealed for 30 min, and incubated overnight at 4 °C in rabbit anti-m⁶A antibody (1:1000, #202003, Synaptic Systems, Germany). After that, the membrane was washed in TBST thrice for 5 min and further incubated for 2 h in corresponding Dylight™ 800 conjugated anti-rabbit IgG (H&L) (GOAT) antibody (1:10,000, #611–145-002, ROCKLAND, USA) at room temperature (~25 °C). After washing thrice again in TBST, the reactive bands were visualised using an Odyssey IR fluorescence scanning imaging system (LICOR, USA).

Total RNA from the isolated hippocampal tissues or HT22 cell samples was extracted using the Total RNApure Kit (#ZP404-1, ZOMANBIO, China) according to the manufacturer’s protocols and quantified using a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific). Further, the m⁶A level was identified using the EpiQuik m⁶A RNA Methylation Colourimetric kit (#P-9005, AmyJet Scientific, USA). Briefly, 300 ng RNA sample with 80 μL RNA high binding solution (BS) was added dropwise to strip wells and incubated for 90 min at 37 °C. After washing thrice with wash buffer (WB), the capture antibody (CA), detection antibody (DA), and enhancer solution (ES) were added sequentially and incubated separately for 1 h at room temperature (approximately 25 °C). After washing five times in WB, the wells were incubated with 100 μL colour-developing solution (DS) and protected from light for ~6 min at which time the solution turned blue in the m⁶A positive wells, then 100 μL stop solution (SS) was added to arrest the reaction. Finally, the absorbance of the stable yellow colour was determined at 450 nm using SoftMax Pro 7 software (SMP7, Molecular Devices, USA). The percentage of m⁶A in the samples was calculated using the following formula: ${\mathrm{m}}^6\mathrm{A}\%=\frac{\left(\mathrm{Sample}\mathrm{OD}-\mathrm{NC}\mathrm{OD}\right)÷300}{\mathrm{PC}\mathrm{OD}-\mathrm{NC}\mathrm{OD}}\times 100\%$ where NC is the negative control and PC is the positive control).