title: GSM8074060: hCSF1_5xFAD_PILRAKO_rep2; Homo sapiens; RNA-Seq
library_construction_protocol: Brains were manually homogenized using a 7mL Dounce homogenizer by adding 4 mL of RPMI 1640 and performing 10 strokes with the “loose” pestle followed by 10 strokes with the “tight” pestle. Samples were then run through a pre-soaked 70uM filter then the filter was washed with 10mL of RPMI 1640. The sample was pelleted by centrifugation and myelin was removed by resuspension in 8L of 30% Percoll overlaid with 2 mL of 1X DPBS centrifuged at 400xg for 20 minutes with acceleration set to 0 and brake set to 4. The myelin band and supernatant were discarded, and cell pellets were resuspended in 70μL MACS buffer (0.5% bovine serum albumin in 1X DPBS) + 30μL Mouse Cell Removal beads (Miltenyi) and incubated at 4°C for 15 minutes. Magnetically labeled mouse cells were separated using LS columns and the MidiMACs separator (Miltenyi) while the unlabeled human cells were collected in the flow through. scRNA-seq library preparation was performed according to the 10X Genomics Chromium Single-cell 3′ Reagents kit v3.1 user guide except that sample volumes containing 26,500 cells were loaded onto the 10X Genomics flow cell to capture ≈10,000 total microglia.