miR-142-3p encapsulated in T lymphocyte-derived tissue small extracellular vesicles induces Treg function defect and thyrocyte destruction in Hashimoto's thyroiditis
The thyroid tissue was very gently dissociated into small pieces and incubated with collagenase D and DNase I for 30 min at 37°C under mild agitation. Then, a filtration step with a 0.70 µm pore size filter is applied to remove the largest elements. The remaining liquid was differentially centrifuged at 300 × g for 10 min and 2,000 × g for 20 min to remove cells and tissue debris. The supernatant was then further centrifuged at 16,500 × g for 20 min and at 118,000 × g for 2.5 h to collect the sEV.   Total RNA from cell or tissue sEV was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The RNA concentration was confirmed using absorbance measurements with a NanoDrop 2100 (Thermo Scientific). A total amount of 1ng-500ng RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using QIAseq miRNA Library Kit (Qiagen, Frederick, MD) following manufacturer's recommendations and index codes were added to attribute sequences to each sample.