GSM8155398: Spleen, WT, replicate 1, scRNAseq; Mus musculus; RNA-Seq
Single-cell suspension of splenocytes was obtained by enzymatic digestion. Cell sorting was performed on FACSymphony S6 sorter (BD Biosciences). To sort different immune cells spiked in equal numbers, the total single-cell suspension was enriched prior to staining and cell sorting by depletion of CD3e+ and CD19+ cells using biotin-labeled monoclonal antibodies and MagniSort Streptavidin Negative Selection Beads (Thermo Fisher Scientific; MSNB-6002-74). CD3e+ and CD19+ cells were sorted from a not enriched fraction of the sample. 1985 T cells (CD3e+), B cells (CD19+), Basophils (CD200R3+, Fcer1+, CD45dim), ILCs (CD3e-, CD127+, CD90.2+), Macrophages (F4/80+, CD64+), NKT cells (CD3e+, CD161+), NK cells (CD3e- CD161+), cDC1s (CD11c+, MHCII+, XCR1+), cDC2s (CD11c+, MHCII+, CD172a+), neutrophils (CD11b+ Ly6G+), monocytes (CD11b+, Ly6G-, Ly6C+), eosinophils (CD11b+ SiglecF+) , pDCs (CD11b- Bst2+ Ly6C+) and 205 Mast cells (Fcer1+, CD117+, CD11c-) were sorted, centrifuged and resuspended in Rhapsody buffer. The sorted single-cell suspension was resuspended at an estimated final concentration of 2000 cells/µl and loaded on a BD Rhapsody cartridge with a targeted capture of 15000 cells. Reverse transcription, cDNA amplification and library construction were performed following the manufacturer's instructions (23-24117(02)). Libraries were sequenced on a NovaSeq6000 flow cell (Illumina).