GSM7817735: CD45+ WT mouse 1 replicate 1_abs 3' CITEseq; Mus musculus; OTHER
To isolate immune cells, after perfusion, brains were digested for 45 min in RPMI containing 10 mM HEPES, 0.5 mg/ml Collagenase Type IV (Sigma Aldrich) and 0.05 mg/ml DNase I (Sigma Aldrich) at 37°C. Cells were then isolated using density gradient centrifugation. The isolated brain cells were directly sorted on an BD Aria III cell sorter for sequencing experiments. The left hemisphere was used to sort CD45+ cells to perform scRNAseq with SeqWell v3 (Aicher et al. 2019). The right hemisphere was used for index sorting of CD3+ cells into 384 well plates (Penter et al. 2018) for scRNAseq with SmartSeq2 (Picelli et al. 2014). Only cells isolated with a purity of > 98% were used for further analysis. CNS CD45+ cells from the brains of B57BL/6 Zfyve26-/- 𝐶𝑋𝐶𝑅4;𝐶𝑟𝑒𝐸𝑅(𝑇2)−𝐼𝑅𝐸𝑆−𝐺𝐹𝑃;𝑅26𝐶𝐴𝐺−𝐿𝑆𝐿−𝑡𝑑𝑇 or Zfyve26+/- 𝐶𝑋𝐶𝑅4;𝐶𝑟𝑒𝐸𝑅(𝑇2)−𝐼𝑅𝐸𝑆−𝐺𝐹𝑃;𝑅26𝐶𝐴𝐺−𝐿𝑆𝐿−𝑡𝑑𝑇 animals were isolated as described above. These cells were labeled with ADT antibodies (Biolegend) according to the manufacturer's protocol for TotalSeq™-A antibodies. 50μL cell suspension with 1x106 cells were resuspended in staining buffer (2% BSA, Jackson Immuno Research; 0.01% Tween-20, Sigma-Aldrich; 1x DPBS, GIBCO) and 5 μL mouse TruStain FcX™ FcBlocking (Biolegend) reagent were added. The blocking was performed for 10 min at 4°C. In the next step 1μg unique TotalSeq-A antibody was added to each sample and incubated for 30 min at 4°C. After the incubation time 1.5mL staining buffer were added and centrifuged for 5min at 350 g and 4°C. Washing was repeated for a total of 3 washes. Subsequently, the cells were resuspended in an appropriate volume of 1x DPBS (GIBCO), passed through a 40 μm mesh (Flowmi™ Cell Strainer, Merck) and counted, using a Neubauer Hemocytometer (Marienfeld). Cell counts were adjusted and hashtagged cells were pooled equally. The cell suspension was loaded, with 25,000 cells to 1.1x105 beads per SeqWell array. RT-transcription cDNA amplification and library generation were performed according to recommendations from Aicher et al. Aicher et al..