scRNA-seq study of hippocampal microglia, perivascular macrophages and perivascular fibroblasts in pre-plaque APP-KI mice
scRNA-seq study of hippocampal microglia, perivascular macrophages and perivascular fibroblasts in pre-plaque APP-KI mice
Cell suspensions were filtered through a 70 μM cell strainer before mixing with Debris Removal Solution. Cells were centrifuged after (300 g for 10 min) and washed with ice-cold FACS buffer (PBS, 2 % FBS, 0.78 mM EDTA). After centrifugation, cells were incubated for 30 min at 4°C with FACS buffer containing Fc block (BD Biosciences) and primary antibody mix. Finally, right before loading the cells on the flow cytometer, cells were stained with DAPI (1:10000).  Single cell suspensions were converted to barcoded Drop-seq libraries by using the Chromium Single Cell 3' Library, Gel Bead & Multiplex Kit and Chip Kit (10X Genomics; Pleasanton, California, USA). cell libraries were prepared using 10X Genomics Chromium Next GEM Single Cell 3' kit v3.1, according to the manufacturer's instructions. As sorted PVMs and fibroblasts were in low abundance, we processed the whole sample, while for microglia cells we aimed for 5,000 cells per sample. Libraries were sequenced using an SP flowcell on an Illumina NovaSeq S6000 instrument, with sequencing parameters recommended by 10X Genomics, aiming for 40,000 reads per cell.