m5C Bis tRNA seq
m5C Bis-tRNA-seq was performed to profile tRNA m5C modifications at single-base resolution. Briefly, total RNA was isolated from young and aged murine BMSCs, followed by sodium bisulfite treatment to convert unmethylated cytosines to uracils. tRNA fragments were size-selected and converted into a sequencing library through reverse transcription and adapter ligation. High-throughput sequencing then allowed for the precise identification and quantification of m5C sites by tracking C-to-T conversions in the aligned sequences.