WT (JK) and CD47- (JINB8) T cells (25x106) were cultured using HITES medium (90% Ham's F-12, 10% RPMI 1640 medium, 5 mM HEPES, 2 mM glutamine, 0.1% BSA, 5 μg/ml insulin, 5 ng/ml sodium selenite, 5 μg/ml transferrin (Thermo Fisher Scientific, Waltham, MA), 200 nM hydrocortisone Sigma Aldrich, St. Louis, MO) for 24-48 h at 37°C. The cells were harvested by centrifugation at 1300 RPM for 5 minutes. Cell pellets were washed with 1X PBS for 5 minutes and used for total RNA extraction. Cell supernatants were collected into new tubes and centrifuged at 395xg (1300 RPM) for 5 minutes. Cell supernatants were collected into new tubes and centrifuged for 10 minutes at 2103xg using using Thermo Sorvall Legend XTR (3000 RPM). The supernatants were concentrated using Centrcon-YM-10 filters to 500 µl. The EVs were precipitated using the Exo-quick kit (SBI System Bioscience, Palo Alto, CA) or Exo-spin™ kit (Cell Guidance Systems, St. Louis, MO ) according to the manufacturer’s instructions. The precipitated EVs were used for isolating total RNA and/or miRNA for real time PCR analysis.
Extracted molecule
total RNA
Extraction protocol
Trizol (Invitrogen) and RNaseazy (Qiagen) kits were used to extract total RNA according to the manufacturer's instructions.
Label
Biotin
Label protocol
Labeling was performed on a sample-by-sample basis according to manufacturer’s guidelines for use with the Affymetrix Mouse HuGene-1_0-st-v1 GeneChips (Affymetrix, Inc). DNase treatment was included as part of isolation to remove possible contaminating DNA. Bioanalyzer nanochip (Agilent, Inc) and NanoDrop (ThermoFisher, Inc) were used to validate and quantitate the RNA prior to cRNA synthesis and labeling. 300 ng of total RNA was used for labeling with theAmbion WT Expression Kit (cat#4411974) and Affymetrix kit (Cat # 900652).After hybridization, the probe arrays were stained with streptavidin-phycoerythrin solution (Molecular Probes) and enhanced using an antibody solution containing 0.5 mg/mL of biotinylated anti-streptavidin (Vector Laboratories).
Hybridization protocol
The hybridization cocktail containing the fragmented and labeled cDNAs were hybridized to Affymetrix mouse GeneChip 1.0 ST microarrays. The microarrays were washed and stained in an Affymetrix Fluidics Station using standard Affymetrix protocols. For microRNAs, 1 microgram of total RNAs was labeled with Affymetrix FlashTag kit using normal affymetrix procedure for labeling, hybridization, staining and scanning.
Scan protocol
Genechips were scanned using an Affymetrix Gene Chip Scanner 3000.
Data processing
Gene expression intensities were calculated using GeneChip Command Console Software (AGCC). .cel files generated by the Affymetrix AGCC program were imported in the Affymetrix Expression Console software and RMA (Robust Multichip Analysis) normalization was performed to generate the .chp files. The .chp files were normalized, log2 transformed, and summarized.
