NextSeq 500 sequencing; Peripheral blood transcriptomic profiles of kidney allograft pathology: biological pathways, comparison with biopsy signals and cellular origin
Peripheral blood sample collection: Blood samples were collected at time of indication biopsy or protocol biopsy at Nantes' hospital, France. They were isolated using density gradient and frozen in their respective serum complemented with 10% DMSO for storage in liquid nitrogen and shipment. Upon arrival in Leuven, Belgium, they were then thawed, centrifuged at 200g for 5 minutes at 4°C before resuspension in PBS containing 0.04% UltraPure BSA (AM2616, ThermoFisher Scientific) and strained through a 40 µm cell strainer to further remove cell clumps and large fragments. Cell number and viability was measured using Luna Cell counter.  Libraries for scRNASeq were generated using the Chromium Single Cell 5′ library and Gel Bead & Multiplex Kit from 10x Genomics. We aimed to profile 5000 cells per library if sufficient cells were retained during the process. All libraries were sequenced on Illumina NextSeq until sufficient saturation was reached. A