GSM7037548: WT, replicate 2, scRNAseq; Mus musculus; RNA-Seq
To retrieve single cell suspensions from spleen, freshly-isolated organs were injected with Collagenase D/DNAse I solution (2mg/ml and 1mg/ml) with a syringe followed by a 15-minute incubation at 37°C. The organs were transferred into GentleMACS C-Tube containing 3ml PBS and subjected to the pre-defined “37 SDK” program in the gentleMACS Octo Dissociator. Retrieved cell suspensions were filtered through a 70 𝜇m filter and cells were pelleted, washed with PBS and subjected to erythrocyte lysis. Splenocytes were enriched for T cells with the MACS T cell isolation kit II (Milteny) according to manufacturer's protocol. Enriched T cells from spleen were subjected to antibody surface staining (B220, CD3, CD11c, TCR𝛿, TCR𝛽) after Fc receptor blockage. The stained cell suspensions were subjected to flow cytometry sorting (Astrios II). After sorting, viable cell numbers were determined and subjected to 10X library preparation (Next GEM Single Cell 5' Lib Kit V1.1) according to the manufacturer's protocol using the 10X Chromium device. The 5'GEX library was consequentially sequenced on a HiSeq platform with 100/8/100 bp and 28/9/91 bp parameters.