title: nCas9(D10A).rep2
design: To evaluate guide RNA-independent off-target RNA editing by the base editor, HEK293T cells were co-transfected with plasmids encoding both the base editor, EGFP and the sgRNA. Seventy-two hours post-transfection, the top 15% EGFP-positive cells were sorted by flow cytometry. The sorted cells were centrifuged, resuspended in 1 ml Trizol (Ambion, 15596018), and then shipped on dry ice to Personalbio (Shanghai) for mRNA extraction, library construction, and sequencing analysis. Paired-end 2150-bp sequencing was performed on the Illumina platform, generating approximately 50 million reads per sample. Raw sequencing data were processed to remove adapters and low-quality reads. Specifically, 3' end adapter sequences were trimmed using Fastp(https://github.com/OpenGene/fastp), and reads with an average quality score below Q20 were discarded.