title: GSM9101815: scRNAseq HTO human microglia 1; Homo sapiens; RNA-Seq
library_construction_protocol: Single cell: At 3 (C9-HRE, C9-ISO) or 6 months (CTRL and C9KO) of age, xenotransplanted mice were euthanized with an overdose of sodium pentobarbital and immediately perfused with ice-cold 1x DPBS (Gibco, Cat. no. 14190-144) supplemented with 5 U of heparin (LEO). After perfusion, one brain hemisphere per mouse was placed in FACS buffer (1X DPBS, 2% FCS and 2 mM EDTA) + 5 µM actinomycin D (ActD; Sigma, Cat. no. A1410-5MG) for transcriptomics. Brains were dissociated using Miltenyi Neural Tissue Dissociation Kit P (Miltenyi, Cat. no. 130-092-628) supplemented with 5 µM ActD. Samples were then passed through a 70-µm strainer (BD2 Falcon), washed in 10 ml of ice-cold FACS buffer with 5 µM ActD and spun at 300g for 15 min at 4 °C. ActD was included during tissue collection and enzymatic dissociation to stop transcriptional activity, and it was stopped following the myelin removal step to minimize toxicity from prolonged exposure. After dissociation, myelin was removed using 30% isotonic Percoll (GE Healthcare, Cat. no. 17-5445-02) solution. Cells resuspended in Percoll were centrifuged at 300g for 15 min at 4 °C. After centrifugation, layers of myelin and cellular debris were discarded and FC receptors were blocked in FcR blocking solution (1:10, Miltenyi, Cat. no. 130-092-575) in cold FACS buffer for 10 min at 4 °C. Cells were then washed in 5 ml of FACS buffer, pelleted and incubated with the following antibodies: PE-Pan-CD11b (1:100, Miltenyi, Cat. no. 130-113-806), BV421-mCD45 (1:100, BD Biosciences, Cat. no. 563890), APC-hCD45 (1:50, BD Biosciences, Cat. no. 555485), Total-Seq A cell hashing antibodies (1:500, BioLegend) and viability dye (1:2.000, eFluor 780, Thermo Fisher Scientific, Cat. no. 65-0865-14), in cold FACS buffer for 30 min at 4 °C. Cells were then washed, pelleted down, resuspended in 500 µl of FACS buffer. Cells were passed through a 35-µm strainer before sorting and loaded into the input chamber of a MACSQuant Tyto Cartridge. Human cells were sorted based on CD11b and hCD45 expression at 4 °C (Fig. S5a). Single cell: The DNA libraries were generated following the manufacturer's instructions (CG000315 Chromium Next GEM Single Cell 3′ Reagent Kits v3.1 Dual Index). Hashtag oligo libraries were prepared according to the manufacturer's instructions (BioLegend, Total-Seq A Antibodies and Cell Hashing with 10x Single Cell 3′ Reagent Kit v3 3.1 Protocol) using 16 cycles for the index PCR.