title: GSM8996331: Xenografted microglia library 5, mRNA-derived cDNA; Homo sapiens; RNA-Seq
library_name: GSM8996331
library_construction_protocol: Human microglia were freshly isolated from the mouse brain of AppNL-G-F mice. In brief, mice were anesthetized with an overdose of sodium pentobarbital and perfused with ice-cold 1x DPBS (+5U heparin). Freshly collected mouse brains were placed in ice-cold FACS buffer (1x DPBS + 2% FCS + 2 mM EDTA) + 5 μM Actinomycin D (ActD, Sigma, Cat#A1410-5MG). After removal of olfactory bulbs and cerebellum, tissue was dissociated using the Miltenyi neural tissue dissociation kit P (Miltenyi, Cat#130-092-628) by means of mechanical and enzymatic dissociation. Cell suspension was filtered and washed with FACS buffer + 5 μM Actinomycin D and spun at 300g for 5 minutes at 4o­ degrees. After washing, cell pellet was resuspended in 30% isotonic Percoll and centrifuged for 5 minutes at 300g to remove cell debris and myelin layers. Pelleted cells were incubated for 10 minutes with FcR blockers (mouse, 0.1 mg ml-1, 500 Miltenyi, #130-092-575; human, 0.1 mg ml-1, Miltenyi, # 130-059-901) on ice. After washing, cell pellet was stained with: PE-PanCD11b (1:50, Miltenyi, Cat#130-113-806), BV421-mCD45 (1:500, BD Biosciences, Cat#563890), APC-hCD45 (1:50, BD Biosciences, Cat#555485), TotalSeqTM-A cell hashing antibodies (1:500, Biolegend) and viability dye (eFluor 780, Thermo Fisher Scientific, Cat#65-0865-14) in cold FACS buffer during 30 minutes at 4°C. Cells were washed in ice-cold FACS buffer and resuspended and filtered on a 35 μm strainer with 500 ml of FACS buffer for sorting. Alive human microglia sorting was done on a MACSQuant Tyto Cartridge and human cells were sorted based on CD11b and hCD45 expression at 4 °C. For single-cell RNA sequencing, 15,000–20,000 human microglia (CD11b+, hCD45+) from each mouse were sorted on the MACSQuant Tyto and diluted to a final concentration of 1,000 cells μl-1. Since all the samples were individually hashed using Total-Seq A cell hashing antibodies, 2,000-4,000 human microglia per animal were pooled and loaded onto the Chromium Next GEM Chip G (PN no. 2000177). The DNA library preparations were generated following the manufacturer's instructions (CG000204 Chromium Next GEM Single Cell 3′ Reagent Kits v3.1). In parallel, the 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. A total of 5 libraries containing 12 biological replicates were sequenced (GAL002, GAL004, GAL005, H1 and H2), targeting a 90% messenger RNA and 10% hashtag oligo library (50,000 reads per cell), on a HiSeq4000 (Illumina) platform with the recommended read lengths by the 10X Genomics workflow.