Comparative non-coding small RNA profiling of extracellular vesicles from porcine oviduct fluid In vivo and oviduct epithelial cells in different in vitro models
Comparative non-coding small RNA profiling of extracellular vesicles from porcine oviduct fluid In vivo and oviduct epithelial cells in different in vitro models
For the in vivo samples, the oviducts were collected from untreated sows slaughtered at the FBN facility and transported on ice to the laboratory within 20 minutes. The oviduct was rinsed with 8 mL PBS for each tube and the fluids from the same animal were pooled. The oviduct fluids were stored at -70 °C until analysis. EVs were isolated from the oviduct fluids using the polyethylene glycol precipitation followed by ultracentrifugation described by Laezer et. al. (2020). For the in vitro samples, the EVs were isolated from the cell culture media. Total RNA was isolated from EV samples using miRNeasy Micro Kit according to the manufacturer's recommendations (Qiagen). Small RNA sequencing libraries were generated from 1 µg of total RNA using the NEXTFLEX Small RNA-Seq Kit v4 with UDIs according to the manufacturer's recommendations (PerkinElmer). The small RNA libraries were quality assessed for expected fragment length using Agilent High Sensitivity DNA kit and 2100 Bioanalyzer (Agilent Technology). 