Total RNA including small RNAs was extracted four hours after electroporation using the RNeasy Plus Mini Kit (QIAGEN GmbH, Hilden, Germany) and the generated samples were sequenced using an Illumina HiSeq-2500. Demultiplexed reads were filtered for ribosomal RNAs, transfer RNAs, mitochondrial rRNAs, and mitochondrial tRNAs. The reads were aligned to the human reference genome (hg19) using STAR (v2.5.2b) and assigned to genes using Subread (v1.5.2). Only uniquely mapping reads that could unambiguously be assigned to a single gene were considered for analysis (Supplementary Table S1).

For miRNA expression quantification, we performed a quality check of the RNA-seq reads with FastQC 31, mapped the short sequences to the human reference genome (hg19) using BWA 32, and calculated raw read counts of mature miRNAs that are known and annotated in miRBase v21 33 (Supplementary Table S2; See Supplementary Materials for details).

Before differential expression analysis, we aggregated read counts of Ensembl identifiers that represent the same gene and discarded genes with less than 5 read counts in any sample to increase power for detecting differentially expressed genes 34,35. Next, we used DESeq2 36 in R version 3.6.3 37 to assess differential expression for protein-coding genes and miRNAs. Then, we performed independent filtering on the results to remove genes that have no or little chance of showing significant evidence (Supplementary Table S3 and S4). Specifically, the independent filtering uses the mean of normalized counts as a threshold to optimize the number of adjusted p-values ≤ 0.05 36. If the normalized expression of a gene was lower than the threshold, it was discarded. The Benjamini-Hochberg method was then used on the set of remaining genes to correct for multiple comparisons 38. Genes with adjusted p-values ≤ 0.05 were regarded as significantly differentially expressed.

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