Transfer of small RNA from extracellular vesicles secreted by primary human airway epithelial cells to Pseudomonas aeruginosa
P. aeruginosa were exposed to extracellular vesicles (EVs) secreted by primary human airway epithelial cells from 3 donors or PBS vehicle control for 6 h. Total RNA was isolated, small RNA libraries were prepared with the QIAseq smRNA kit (Qiagen) and 75-bp single-end reads were generated using Illumina MiniSeq. To assess transfer of human EV small RNAs to P. aeruginosa, sequences that did not map to the PA14 reference genome were aligned to the human genome. Overall design: P. aeruginosa (1.5*E+07 CFU) were exposed to PBS vehicle control or EVs (5E+09 EV/ml) for 6 h. To avoid possible carry-over of EVs (and miRNA) attached to the outside of the bacteria, the bacteria were washed with PBS and the bacterial outer membrane was lysed with EDTA to release periplasmic contents and factors associated with the bacterial outer membrane and periplasm (77-79). The cells were then washed again in PBS, and cytoplasmic RNA was harvested after lysis of bacterial cell wall and inner membrane. Total RNA was isolated with the miRNeasy Mini Kit (Qiagen, Germantown, MD, Cat. # 217004), including the on-column DNA digestion. Sequencing libraries were prepared with the QIAseq smRNA kit (Qiagen) and 75-bp single-end reads were generated using an Illumina MiniSeq. Sequences that did not map to the PA14 reference genome were aligned to the human genome using CLC Genomics Workbench software (Qiagen).