GSM8578118: B cells, Th17 EAE model, animal 2 [VDJ-A2]; Mus musculus; RNA-Seq
Recipient mice, which had been on a high score for several days to allow for continued immune cell infiltration, were sacrificed and perfused through the left cardiac ventricle with PBS. Brains were dissected by removing the skull using forceps and scissors, while spinal cords were flushed out from the bone column using a 21 gauge needle attached to a syringe filled with PBS. In addition, the vertebral column was opened to collect parts of meninges, which were still connected to the vertebrae. Then, brains and spinal cords were cut into pieces and digested for 30 min at 37 °C with DNase I (Sigma-Aldrich or Roche) at a concentration of 1 mg/ml and collagenase D (Roche) at a concentration of 3.75 mg/ml. Subsequently, the tissues were mashed and passed through a 70 μm cell strainer to prepare a single-cell suspension, followed by centrifugation at 350 g for 10 min at 4 °C. The cell pellet was resuspended in 5 ml 70 % Percoll (Cytiva), which was prepared by mixing 30 ml Percoll with 18 ml Percoll Mix Solution (mixture of 90 ml 10X PBS and 264 ml H2O). Then, the cell suspension was slowly and carefully overlaid with 5 ml 37 % Percoll, which was prepared by diluting the 70 % Percoll solution with 1X PBS, followed by centrifugation at 650 g for 30 min at 21 °C. Then, mononuclear cells were collected from the interface between the 70 % and 37 % layer and washed twice with T cell medium to remove residual Percoll. Finally, cells were either stimulated for intracellular cytokine staining or stained directly for flowcytometric analysis. For each mouse, B cells from CNS, spleen and cLNs were pooled (90.000 B cells in total), centrifuged and, after a washing step, resuspended in PBS containing 0.04 % BSA at a concentration of 1000 cells/μl. The viability of cells was confirmed by staining with trypan blue solution. Further processing of samples was performed using the 10x Chromium Single Cell 5` Solution (Chromium Next GEM Single Cell V(D)J v1.1 with Feature Barcoding Technology for Cell Surface Protein, 10x Genomics) according to the manufacturer`s instructions. Prior to loading on the chip, 5000 cells of each compartment were labeled with anti-mouse hashtag oligonucleotide (HTO) antibodies (TotalSeq™-C0301/C0302/C0303, Biolegend) and pooled together. cDNA libraries were pooled and send for sequencing. scRNA-seq and scBCR-seq libraries were sequenced on NovaSeq S4 Flowcells using 150-bp paired-end reads and 8 bp for the i7 index, aiming for 50,000 reads per cell for gene expression and 5,000 reads per cell for BCR enrichment. Cell hashing libraries were sequenced on NovaSeq S1 Flowcells using 50-bp paired-end reads and 8 bp for the i7 index, aiming for 10,000 reads per cell.