RNA Cas9-independent off-target (base editing)
or RNA sequencing with poly(A) selection, total RNA was quantified using a Qubit 2.0 Fluorometer (Life Technologies), and RNA integrity was assessed using the Agilent TapeStation 4200 (Agilent Technologies). ERCC RNA Spike-In Mix (Thermo Fisher Scientific) was added to normalized RNA samples following the manufacturers protocol.RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs), following the manufacturers instructions. Briefly, mRNA was enriched using oligo(dT) beads, then fragmented at 94C for 15 minutes. First- and second-strand cDNA synthesis was performed, followed by end repair, 3' adenylation, adapter ligation, index addition, and PCR enrichment. Libraries were validated using the Agilent TapeStation and quantified using the Qubit 2.0 Fluorometer and quantitative PCR (KAPA Biosystems).Prepared libraries were multiplexed and sequenced on an Illumina NovaSeq X Plus system (Illumina) using 2150bp paired-end reads. Image analysis and base calling were performed using NovaSeq Control Software (NCS), and raw BCL files were converted to FASTQ format and demultiplexed using bcl2fastq v2.20, allowing one mismatch in index sequences.
SUB15709406
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