Peripheral blood sample preparation

Whole blood was collected from a healthy donor into EDTA tubes (Becton, Dickinson & Co). Separation of PBMC was performed with Ficoll-Paque PREMIUM (GE Healthcare) according to the manufacturer’s instructions. Briefly, for density gradient separation, 4 mL of blood was added to 3 mL of Ficoll-Paque PREMIUM and centrifuged at 400 × g for 30 min at 18 °C without brake. PBMC layer was recuperated and washed twice with PBS at 400 × g for 15 min at 18 °C. PBMC were resuspended in freezing solution (10% DMSO, 90% non-inactivated FBS) and frozen by gradually decreasing temperature (1 °C/min) to –80 °C (cryopreserved). After storage for one week at –80 °C, the cryopreserved sample was rapidly thawed in a water bath in continuous agitation and placed into 25 mL of cold 1× HBSS. Cells were washed once in ice-cold 1× HBSS and resuspended in DMEM before sorting. To avoid batch effects, freshly isolated PBMC were sorted in parallel with the cryopreserved material and distributed over the same sequencing pools. Therefore, blood from the same donor was isolated as described above and directly resuspended in DMEM. Dead and damaged cells were identified by propidium iodide staining.