Single cell sequencing of astrocytes in the APP NL-F and APP PS1 models of ß-amyloidopathy
Single cell sequencing of astrocytes in the APP NL-F and APP PS1 models of ß-amyloidopathy
Mice (3 per age/genotype combination) were anesthetised using isoflurane and decapitated. CO2 was not used in order to avoid confounding effects on gene expression. The neocortices were dissected and placed in ice cold Dulbecco's phosphate-buffered saline (DPBS) (ThermoFisher). Tissue was enzymatically and mechanically dissociated using the Adult Brain Tissue Dissociation Kit, as per the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, tissue was combined with the enzyme cocktails. The gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec) was used to mechanically dissociate the tissue at 37oC for 30 minutes (program 37C_ABDK_01). The digested tissue was filtered (70 μm) with DPBS. Myelin and cell debris were removed using the Debris Removal Solution and centrifugation steps. Erythrocytes were removed using the Red Blood Cell Removal Solution and centrifugation steps. Cells were transported to the FACS facility in PBS containing 0.5% bovine serum albumin. FACS of astrocytes was carried out by the University of Edinburgh FACS facility on a BD Aria II. Gates were set up to gate out debris and dead cells, along with clumps and auto fluorescent cells. GFP positive cells were collected. 10x Genomics sample preparation was done using the 10x Genomics Single Cell 3' Reagent kits V3 (10x Genomics, Pleasanton, California) according to the manufacturer's instructions. Incubation of the GEMs produces barcoded cDNA from poly-adenylated mRNA. GEMs were then broken and pooled fractions are recovered. Silane magnetic beads were used to purify the first-strand cDNA from the post GEM-RT reaction mixture, which includes leftover biochemical reagents and primers. Barcoded, full-length cDNA was amplified via PCR to generate sufficient mass for library construction.