Analysis of RNA off-target activities with RNA sequencing

For transcriptome analysis, base editors were co-transfected with sgRNA-1 for 48 h. And GFP/mCherry double-positive cells were isolated using a Moflo XDP (Beckman Coulter) and total RNA was isolated using Trizol reagent for RNA-seq. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries with standard Illumina protocols. RNA-seq was performed using Illumina HiSeq X Ten platform in Shanghai Novelbio Ltd. For each library, ~50–60 million reads were generated. We applied the reads filtering towards the raw reads after sequencing to achieve the clean data following the criteria: a) 10% base quality <15 b) 13% base quality <20. RNA sequencing data was aligned to the human reference genome (GRCh38) using STAR (v2.5.2b). Variants were called using the GATK best practices pipeline using Picard and GATK 3.8. Single-nucleotide variants (SNVs) were filtered to include loci with reads >10 and labeled as A–G or T–C for to evaluate RNA off-target activities.