GSE168187
miRNA sequencing using Jurkat and JinB8 T cell line (CD47-deficient) and their Extracellular vesicles (EVs)
Extracellular vesicles (EVs) mediate cell-cell communication including the intercellular transfer of miRNAs, which alter gene expression in target cells. Previously, we have shown that CD47 is also present on EVs and alters their effects on target cells, suggesting that specific surface markers define functionally distinct EVs. This hypothesis will be addressed by comparing total cellular versus EVs miRNA contents between Jurkat and JinB8 (CD47 deficient) T cells. The Jurkat T cell line (E6.1) was purchased from ATCC. The cells were maintained using RPMI 1640 containing 10% FBS, glutamine, penicillin, and streptomycin (Gibco). The Jurkat and JinB8 T cells used for experiments were maintained less than 6 weeks in culture. The cultured Jurkat were washed with IXPBs followed by a second wash with HITES medium (DMEM/F12 supplemented with, bovine serum albumin, hydrocortisone, insulin, transferrin, and trace elements). EVs were isolated from Jurkat T cells were cultured overnight using HITES medium. The conditioned media from Jurkat T cells were collected, and cell debris were removed using centrifugation at 300 g for 5 minutes and 2500 g for 10 minutes respectively. Centrifuged media were concentrated using Ultra-4 centrifugal filters to about 1 ml volume and further centrifuged at 10,000 g for 10 minutes. EVs were isolated using an Exo-Quick kit (SBI). miRNaseazy (Qiagen) kits were used to extract total RNA from Jurkat and JinB8 T cells and their EVs according to the manufacturer's instructions. Overall design: Jurkat and JinB8 cells were cultured using serum-free HITES media and EVs were extracted. RNA was extracted using miRNA easy kit from Qiagen