Comprehensive miRNA Profiling from Cell Cultures

Total RNA purification from cell cultures was performed by miRNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. RNA concentration was determined with a NanoDropOne spectrophotometer (Thermo Fisher, Waltham, MA, USA) and its quality was assessed with the TapeStation 4200 (Agilent Technologies, Santa Clara, CA, USA). Indexed libraries were prepared from 500 ng/each purified RNA using the NEXTFLEX Small RNA-Seq Kit v3 (PerkinElmer). Libraries were quantified with the TapeStation 4200 (Agilent Technologies) and Qubit fluorometer (Invitrogen Co.), then pooled such that each index-tagged sample was present in equimolar amounts. The pooled samples were then subjected to cluster generation and sequencing using an Illumina NextSeq 550 Dx System (Illumina) in a 1 × 75 single-end format. The generated raw sequence files (.fastq files) underwent quality control analysis using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) (accessed on 22 September 2022). The sRNAbench tool [23 (link)] was then used to remove adapter sequences and low-quality reads to obtain the miRNA expression profiles. Finally, the Bioconductor package DESeq2 [24 (link)] in R was used to normalize the data using the median of ratios method, and to perform the differential expression analysis between the various experimental condition.