title: DNBSEQ-G400 paired end sequencing; Effects of prostate cancer patient urinary extracellular vesicles and hypoxic extracellular vesicles from prostate cancer cell lines on the transcriptomes of cancer-associated and normal fibroblasts
design: Effects of prostate cancer patient urinary extracellular vesicles and hypoxic extracellular vesicles from prostate cancer cell lines on the transcriptomes of cancer-associated and normal fibroblasts
library_construction_protocol: Cells were seeded in DMEM-F12 complete medium at a density of 1x105 cells per well in 12-well plates and 1x104 EVs per cell (total 1x109 EVs / well) were added to the cells and they were grown as a monolayer for 48 hours. Untreated control cells were grown in parallel. After that, the cells were washed with PBS and lysed with 1ml TRI-Reagent (Sigma Aldrich, USA) and RNA was extracted according to manufacturers protocol. The cells were washed with PBS and lysed with 1ml TRI-Reagent (Sigma Aldrich, USA) and RNA was extracted according to manufacturers protocol. Briefly, 200 l of chloroform was added, the samples were centrifuged at 12 000g for 15 min at +4C, the aqueous phase was transferred to a new tube and isopropanol was used to precipitate the RNA by centrifuging at 12 000g for 10 min at +4C. After that, the RNA was washed with 70% Ethanol-DEPC and the DNA was removed with DNA-free (Thermo Fisher Scientific, USA) DNA removal kit according to manufacturer's protocol. The RNA concentration was measured with NanoDrop 1000 (Thermo Ficher Scientific, USA) and the integrity of the RNA was measured with Agilent Bioanalyzer using RNA Pico chip (Agilent Technologies, USA).  For transcriptome sequencing, the rRNA was removed from 200 ng of total RNA using MGIEasy rRNA depletion kit (MGI, China). The transcriptome libraries were built using MGIEasy RNA Directional Library Prep Kit (MGI, China) according to manufacturers protocol. Briefly, the RNA was fragmented into 250 bp pieces, and it was reverse transcribed, and the second strand was synthesized. Adapters were ligated according to the manufacturers recommendations and the product was amplified with PCR.