Monoclonal RPE1(Cas9) cells were harvested at 90% confluency and total RNA was purified using the QIAGEN RNeasy Plus Mini Kit (Cat No.: 74134), according to the manufacturer's protocol. RNA was stored at -80°C and analyzed by RNA-Seq in quadruplicates. For library preparation, total RNA was quantified using the Qubit 2.0 fluorometric assay (Thermo Fisher Scientific). Sequencing libraries were prepared from 125 ng of total RNA using a 3'DGE mRNA-seq research grade sequencing service (Next Generation Diagnostics srl) which included library preparation, quality assessment and sequencing on a NovaSeq 6000 sequencing system using a single-end, 100 cycle strategy (Illumina Inc.) (46 (link)). The bioinformatics workflow included analysis of raw data by Next Generation Diagnostics srl proprietary 3'DGE mRNA-seq pipeline (v2.0) which involves a cleaning step by quality filtering and trimming, alignment to the reference genome and counting by gene (47–49 (link)). We filtered out all genes having < 1 cpm in less than n_min samples and Perc MM reads > 20% simultaneously. Differential expression analysis was performed using edgeR (Supplementary Table S7) (50 (link)).