Single-cell discovery of m⁶A RNA modifications in the hippocampus

Detection of m⁶A with bulk RNA-seq in mouse hippocampus. (A) Schematic diagram of bulk RNA-seq in the mouse brain. The Egfp-Apobec1-Yth is packaged into AAV viruses to infect brain cells. EGFP-positive cells are isolated from the hippocampus and processed for C-to-U edit and m⁶A site identification. (B) Confocal image of mouse hippocampus after AAV infection. Representative image of E-YTH is shown. Half-brain image: Scale bar, 1 mm. Hippocampus image: Scale bar, 400 μm. (C) Number of overlapping C-to-U editing events identified by RNA-seq in hippocampus following Egfp-Apobec1-Yth and Egfp-Apobec1-Yth^mut AAV virus injection and EGFP FACS sorting. Editing events identified in at least two replicates were considered for downstream analyses. n = 3, (Rep) Biological replicates from different animals. (D) Pie chart showing m⁶A localization identified by E-Yth in mouse hippocampus. (TTS) Transcription termination site. (E) Metagene analysis showing C-to-U edit scaled density 500 nt 5′ and 500 nt 3′ from stop codon (0 nt) in E-YTH, E-YTH^mut, E-APOBEC1, and wild-type (WT) samples. The peak value for E-YTH is 2716 editing events. (F) Metagene analysis showing m⁶A density 500 nt 5′ and 500 nt 3′ from stop codon (0 nt). m⁶A sites were obtain after eliminating background from E-YTH editing sites. m⁶A peak density occurs 118 nt downstream from the stop codon. (G) Histogram of m⁶A site counts over mutation per read (m/k) ratio. Minimum threshold: 5%.