RNA-Seq of human: 293T cells
For analyzing RNA off-target across the transcriptome, each selected sample was made up of two biological repeats, and total RNA was extracted from two biological repeats using the TRIzol reagent (Invitrogen), respectively. RNA library preparation was performed using the TruSeq Stranded Total RNA Library Prep Gold kit (Illumina) with an initial input of ~500 ng extracted RNA per sample, using SuperScript III (Invitrogen) for first-strand synthesis. Depletion of ribosomal RNA (rRNA) was confirmed after the initial rRNA removal step by fluorometric quantification using the Qubit RNA HS Assay kit (Invitrogen). IDT for Illumina TruSeq RNA UD Indexes (96 indexes) were used to barcode each library with unique dual indexes to mitigate index hopping. RNA-seq libraries were examined on a high-resolution QIAxcel (Qiagen) and pooled on the basis of qPCR quantification with the KAPA Library Quantification Kit Illumina (KAPA Biosystems) or the NEBNext Library Quant Kit for Illumina (NEB).