RNA-seq of Equus caballus: uterine extracellular vesicles
RNA was isolated from extracellular vesicles (EVs) using the miRNeasy micro kit (QIAGEN) according to the manufacturers instructions. RNA concentration was measured by different RNA quantification methods: Agilent RNA 6000 Pico assay (Agilent 2100 Bioanalyzer, Agilent Technologies Schweiz AG, Basel, Switzerland) for RNA quantity and quality profiles of EVs samples; and (2) Quantus Fluorometer (Promega AG, Dbendorf, Switzerland) together with QuantiFluor RNA System kit (Promega).RNA-Seq library preparation was performed starting from 27 ng total RNA by using the SEQuoia Complete Stranded RNA Library Prep Kit (Bio-Rad Laboratories AG, Cressier, Switzerland) following manufacturers instructions. To deplete ribosomal RNA fragments, QIAseq FastSelect depletion kit (QIAGEN AG) was used in the first step of the library preparation. Sequencing of the 26 libraries was conducted on an Illumina NovaSeq 6000 instrument at the Functional Genomics Center Zurich (FGCZ). Pooled barcoded libraries were run on one SP flow cell. Paired-end sequencing was performed with 92 bp for read one (cDNA insert) and 8 bp for read 2 (UMI sequence for removal of PCR duplicates).