To apply mechanical stretch to the cells, 0.35-0.45 x 106 cells in 2 ml of medium were seeded onto each well of the 6-well Bioflex multiwall Plates (Flexcell International, Hillsborough, NC, USA) precoated with collagen-1. Monolayers were maintained for an additional 24 hour until reached approximately 80% confluency, and after fresh medium was replaced, plates were mounted in a Flexcell FX-4000 Stretch Unit (Flexcell International). Regimens of 5% continuous stretch or 10% cyclical stretch, at intervals of 40 cycles/min for 24 hours, were used. Cells grown on non-stretched membranes were treated in an identical manner and served as controls.
Growth protocol
MLE12 cell line was obtained from American Type Culture Collection (ATCC) (Manassas, Virginia). This is a murine cell line that expresses features of type II lung epithelial cells. The cells were cultured in DMEM/F12 supplemented with insulin (0.005 mg/ml), transferrin (0.01 mg/ml), sodium selenite (30 nM), hydrocortisone (10 nM), β-estradiol (10 nM), HEPES (10 mM), L-glutamine (2 mM) and extracellular-depleted 2% bovine serum.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from 20 µg of EVs using Total Exosome RNA and Protein Isolation Kit (Invitrogen, cat. no. 447845) following the instruction manual. Total RNA from 3.5 x 106 cells was isolated using Quick-RNA Mini Prep (Zymo Research) according to manufacture provided protocol. RNA integrity was tested by agarose gel electrophoresis analysis. RNA quality and concentration was measured by NanoDrop Spectrophotometer.
Label
biotin
Label protocol
FlashTag™ Biotin HSR Ligation 1. Briefly microfuge the 15 μL of tailed RNA and place on ice. 2. Add 4 μL 5X FlashTag Biotin HSR Ligation Mix (Vial 5) to each sample. 3. After the Ligation Mix has been added, add 2 μL of T4 DNA Ligase (Vial 6) to each sample. Do not make a master mix at this step, as auto-ligation can occur. 4. Mix gently (do not vortex) and microfuge. 5. Incubate at 25°C (room temperature) for 30 minutes. 6. Stop the reaction by adding 2.5 μL HSR Stop Solution (Vial 7). Mix and microfuge the 23.5 μL of ligated sample. 7. Remove 2 μL of the biotin-labeled sample for use with the ELOSA QC Assay (Appendix A). It is acceptable to store the 2 μL of biotin-labeled sample on ice for up to 6 hours or at –20°C for up to 2 weeks, and run the ELOSA QC Assay at a convenient time. If the ELOSA QC Assay is not performed, it is recommended that 2 μL of biotin-labeled sample be saved until the array QC is complete. Retaining this sample will enable one the ability to troubleshoot possible target preparation issues, if needed. 8. The remaining 21.5 μL biotin-labeled sample may be stored on ice for up to 6 hours, or at –20°C for up to 2 weeks, prior to hybridization on GeneChipTM miRNA Arrays or miRNA Array Plates.
Hybridization protocol
Hybridization 1. Bring the reagents listed in Step 3, below, to room temperature. 2. Completely thaw and then heat the 20X Eukaryotic Hybridization Controls (bioB, bioC, bioD, cre from GeneChipTM Eukaryotic Hybridization Control Kit) for 5 minutes at 65°C. 3. Add the following components in Table 3.1 to the 21.5 μL biotin-labeled sample in the order listed, to prepare the array hybridization cocktail: 4. Incubate at 99°C for 5 minutes, then 45°C for 5 minutes. 5. Aspirate 100 μL (400/169 format array) or 130 μL (100 format array) and inject into an array 6. Remove the pipet tip from the upper right septum of the array. 7. Cover both septa with 1/2 Tough-SpotsTM to minimize evaporation and/or prevent leaks. 8. Place the arrays into hybridization oven trays. 9. Load the trays into the hybridization oven. 10. Incubate the arrays at 48°C and 60 rpm for 18 hours Table 3.1 Hybridization Cocktail (for a single reaction) Component Volume for a 400/169 Format Array (miRNA 1.0 and 2.0 Arrays) Volume for a 100 Format Array (miRNA 3.0 and later designs) Final Concentration 2X Hybridization Mix 50 μL 66 μL 1X 27.5% Formamide (Vial 12) 15 μL 19.2 μL 4% DMSO 10 μL 12.8 μL 9.7% 20X Hybridization Controls 5 μL 6.6 μL 1X Control Oligo B2, 3nM 1.7 μL 2.2 μL 50 pM Nuclease-free Water N/A 3.7 μL Total Volume 81.7 μL 110.5 μL Component 400/169 Format Array 100 Format Array (miRNA 1.0 and 2.0 Arrays) (miRNA 3.0 and later designs) Biotin-labeled Sample 21.5 μL 21.5 μL Hybridization Master Mix 81.7 μL 110.5 μL
Scan protocol
Fluidics Station 450 Protocol FS450_0003 (400/169 format array,Arrays were scanned with an Affymetrix 3000 7 G scanner
