GSM7312152: Bone marrow, Stroke, ScRNAseq, rep1; Mus musculus; RNA-Seq
Three mouse single-cell RNA experiments were performed in this study: one on a pool of myeloid cells isolated from the heart, lung, liver, spleen and blood; and the other two on bone marrow myeloid cells, either from stroke and control mice, or from naïve recipients after BM transplantation. For the first experiment, mononuclear cell suspensions from peripheral organs were incubated with anti-CD16/CD32 antibody to block nonspecific binding and stained for CD45+CD11b+ myeloid cells (see antibodies list). Five unique cell hashtags antibodies were also used to label cells from each organ. 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, lung, liver, spleen and blood from the same animal were pooled together in the same collecting tube (cell ratio of 1:4:4:4:4, respectively). After sorting, all cells were centrifuged and cautiously resuspended to a final concentration of 1000 cells/l. Cells were then transferred to the Next GEM chip according to manufacturer's instructions. ScRNA-seq and cell hashing libraries were prepared using the 10x Chromium Single Cell 3′ Solution combined with cell hashing, as per established protocols.10    For the second and third experiments, bone marrow cells suspensions were stained with a cocktail of antibodies against markers of the lymphoid lineage (CD3, CD4, CD8a, CD19 and Ter119) and mature neutrophils (Ly6G, see antibodies list). Immediately prior to cell sorting, PI or 7-AAD were also added to label dead cells. Cells were sorted according to the surface markers (SH800S Cell Sorter, Sony Biotechnology, or FACS Aria II Fusion cell sorter, BD Biosciences, Inc.) and the negative fraction of BM myeloid cells (depleted of lymphoid lineage cells and neutrophils) was collected. After sorting, cells were centrifuged and cautiously resuspended to a final concentration of 1000-1200 cells/l. Cells were then further processed according to manufacturer's (10X) instructions. The scRNA-seq libraries were prepared using the 10x Chromium Single Cell 3′ Solution.   In all three experiments, 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 1000 using 20,000 reads per cell. Cell-surface protein expression libraries were sequenced on an Illumina NextSeq 1000 aiming for 5,000 reads per cell. Libraries were performed according to the manufacter's instructions (single cell 3' v3.1 protocol and single cell 3' v3.1 with feature barcoding technology for Cell surface protein, 10x Genomics).