title: GSM6703379: Library 23 cell hashing; Homo sapiens; RNA-Seq
library_construction_protocol: Human microglia were freshly isolated from the mouse brain of AppNL-G-F, Apphu/hu or AppNL-G-FApoE-/-  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. Of note, 5 μM Actinomycin D was kept during collection, dissociation and washing and was removed afterwards to avoid cellular toxicity. 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 mouse FcR blockers (1:10 in FACS Buffer, Miltenyi, Cat#130- 092-575) 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. In this manuscript, we generated a total of 20 libraries containing 95 biological replicates for single cell RNA sequencing. In brief, we sorted 15000-20000 alive human microglia (CD11b+, hCD45+) per mouse and adjusted the final concentration to 1000 cells/µl. Out of each mouse, 2000 sorted cells were loaded into the Chromium Next GEM Chip G (PN #2000177). Since all the individual replicates were hashed using TotalSeqTM-A cell hashing antibodies, we pooled different samples into the same library. Libraries were prepared using manufacturer's instructions (CG000204 Chromium Next GEM Single Cell 3' Reagent Kits v3.1). For hashtag oligo libraries, we followed manufacturers' instructions (BioLegend - TotalSeq™-A Antibodies and Cell Hashing with 10x Single Cell 3' Reagent Kit v3 3.1 Protocol) using 16 cycles for the index PCR. Libraries were sequenced on a HiSeq4000 or NovaSeq6000 (Illumina) platform with the recommended read lengths by 10X Genomics workflow targeting 90% mRNA and 10% hashtag library (50,000 reads/cell).