RNA-Seq of pig extracellular vesicles  in uterinal fluid: sample 2 of day 9 of estrus
A total amount of 20 ng RNA per sample was used as input material for the RNA sample preparations. Firstly, ribosomal RNAwas removed by Epicentre Ribo-zero rRNA Removal Kit (Epicentre, USA), and rRNA free residue was cleaned up by ethanol precipitation. Subsequently, sequencing libraries were generated using the rRNA-depleted RNA by NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, USA) following manufacturers recommendations. Second strand cDNAsynthesis was subsequently performed using DNA Polymerase I and RNase H. In the reaction buffer, dNTPs with dTTP were replaced by dUTP. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3 ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization.