GSM8172200: Recipient bone marrow, Control donor, ScRNAseq, rep2, CSP; Mus musculus; OTHER
Mononuclear cell suspensions from the heart and the blood were incubated with anti-CD16/CD32 antibody to block nonspecific binding and stained for CD45+CD11b+ myeloid cells. Two unique cell hashtags antibodies were also used to label cells from blood and heart independently. All surface antibodies and hashtag antibodies were incubated for 30 min at 4ºC. Immediately prior to cell sorting, PI was added to all samples to label dead cells. Cells were sorted according to their surface markers (SH800S Cell Sorter, Sony Biotechnology). Sorted and organ-specific labeled cells from the heart and blood from the same animal were pooled together in the same collecting tube. After sorting, all cells were centrifuged and cautiously resuspended to a final concentration of 500 cells/ul. Cells were then transferred to the Next GEM chip according to manufacturer's instructions. ScRNA-seq and cell hashing (cell surface protein) libraries were prepared using the 10x Chromium Single Cell 3′ Solution combined with feature barcoding technology for Cell surfacte protein, as per established protocols. Quality control of all cDNA samples was performed with a Bioanalyzer 2100 (Agilent Technologies) and libraries were quantified with the Qubit dsDNA HS kit. Gene expression libraries were sequenced on an Illumina NextSeq 2000 using 20,000 reads per cell. Cell-surface protein expression libraries (cell hashtags) were sequenced on an Illumina NextSeq 2000 aiming for 5,000 reads per cell. Libraries were performed according to the manufacter's instructions ( single cell 3' v3.1 with feature barcoding technology for Cell surface protein, dual index, 10x Genomics).