Extraction and Analysis of miRNA and RNA from T Cell-Derived Extracellular Vesicles

MicroRNA and total RNA were extracted from WT and CD47⁻ T cell-derived EVs (isolated and purified as indicated in extended methods) using the miRNA easy kit from QIAGEN. The concentration and RNA quality were measured using a RNA-Bio analyzer. 100–200 ng of total RNA was used for first strand miRNA synthesis (Quanta Bioscience) as described (Kaur et al. 2016 (link)). First strand cDNA synthesis used a Maxima kit (2-Step RT PCR, Thermo Scientific) according to the manufacturer’s instructions or from Maxima first strand (Thermo scientific Fisher). U6 was used as a reference control gene for real -time PCR analysis as indicated in the legends. Cytoplasmic and nuclear RNA were extracted using the Cytoplasmic and Nuclear RNA Purification Kit from Norgen BioTek Corporation (Thorold ON). RNA was extracted according to manufacturer’s instructions. CD3⁺ cells from WT and Cd47 null mice (n = 3) were purified using Pan T Cell Isolation Kit II, mouse from Miltenyi Biotec, and mRNA isolation and real-time PCR were performed as described above using primers listed in Figure S7.