PrE and PrS cells were cultured to 70% confluency, conditioned media was collected and EVs were isolated by differential ultracentrifugation, as described above. PrE and PrS cells were lysed, and RNA was collected using QIAzol (Qiagen, Germany). Tissue slices were homogenized in QIAzol using the BeadRuptor 4 (OMNI International, Bedford, NH). RNA was isolated from all intracellular samples with the miRNeasy Mini Kit (Qiagen, Germany) and from EVs using the Serum/Plasma miRNeasy Kit (Qiagen, Germany). MiR quantification from all samples was completed with microRNA Qubit (Thermo Fisher Scientific, Waltham, MA).
MiR libraries were generated from 100 to 150 ng of cell using the QIAseq miRNA Library Kit according to the protocol (Qiagen, Germany). Library quality control and concentrations were determined using a High Sensitivity D1000 ScreenTape with TapeStation Analysis Software A.02.02 (Agilent Technologies, Santa Clara, CA) Libraries (10 µM) were submitted to the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana‐Champaign, where they were titrated for equal loading using the MiSeq followed by sequencing on a HiSeq 4000 with 100 nucleotide forward single reads.
MiR libraries were generated from 100 to 150 ng of cell using the QIAseq miRNA Library Kit according to the protocol (Qiagen, Germany). Library quality control and concentrations were determined using a High Sensitivity D1000 ScreenTape with TapeStation Analysis Software A.02.02 (Agilent Technologies, Santa Clara, CA) Libraries (10 µM) were submitted to the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana‐Champaign, where they were titrated for equal loading using the MiSeq followed by sequencing on a HiSeq 4000 with 100 nucleotide forward single reads.