GSM8653306: S0, replicate 1, scRNAseq; Mus musculus; RNA-Seq
Mice were anesthetized with Pental (1:1 in PBS) and perfused with cold PBS. Whole mouse brains were isolated in intact condition and incubated for 40 min at 37 °C with gentle shaking in the digestion solution (3 ml HBSS /per brain containing 2% BSA, 5 mM EDTA, and 0.2 mg/ml Collagenase type 2 (Worthington)). After the incubation leptomeningeal cells were washed out from the surface of mouse brains with pipetting of digestion solution on the surface and collected in tubes with cold PBS after passing a 100-µm mesh filter. Collected cells were centrifuged at 400 g at 4 °C for 5 min and re-suspended with cold FACS buffer containing 2% FBS and 1 mM EDTA based on PBS w/o Ca2+ and Mg2+ for antibody staining. Intact brains were further digested for parenchymal cell isolation. Brains were dissected in 1 ml of digestion solution containing 2% BSA, 1 mg/ml collagenase D (Sigma) and 1 mg/ml DNase1 (Sigma), and incubated for 20 min at 37 °C. In the middle of incubation, brains were homogenized by pipetting. After the incubation, the homogenate was filtered through a 100-µm mesh and centrifuged at 970 g at 4 °C for 5 min. To enrich brain cells, the cell pellet was re-suspended with 3 ml of 40% Percoll solution and centrifuged at 970 g, w/o acceleration and brake, at room temperature for 15 min. After the centrifugation, the myelin layer was removed, and the rest of the supernatant and cell pellets were washed in cold PBS. Cells were re-suspended with cold FACS buffer for antibody staining for sorting. Cells were kept on ice during all steps, except for the enzymatic digestion and cells for bulk-RNAseq and scRNAseq were processed always with actinomycin D (ActD) to prevent transcriptomic changes, as reported. A derivation of MARSseq protocol was used to prepare libraries for RNAseq. For the single-cell RNA sequencing (scRNAseq) library preparation, cells were isolated from five one-year-old Ms4a3cre: R26-TdTomfl/fl: Cx3cr1gfp  mice and sorted in a 384-well plate with lysis buffer and poly-T barcoded primers and placed at -80C for future processing. When thawed, they are heated to bind the primer to the mRNA and the first strand mix is added for the cDNA-mRNA hybrid. ExoI is added to remove excess primers and all samples are then pooled followed by second-strand synthesis. Pooled samples are amplified overnight using IVT, followed by RNA fragmentation and ligation of the adapter for pooled sample identification. Finally, first strand synthesis and PCR for final sample amplification. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument. RNaseq libraries were sequenced using the Illumina NextSeq 500 with 90 reads, 75 for read 1, which contained the ligation adapter sequence and mRNA sequence, and 15 for read 2, which contained the single sample or single cell barcode and UMI sequences.