GSM8413154: in vivo Daxx WT, KO microglia TAM 3dpi, 1wpi, 8wpi, scRNAseq_mixed sample; Mus musculus; RNA-Seq
Microglia from mice brains were isolated using the Adult Brain Dissociation Kit from Miltenyi Biotech (Bergisch Gladbach, Germany) according to the manufacturer's guidelines. In brief, after euthanasia, mice brains were mechanically and enzymatically digested at 4 oC. Fully digested tissue suspensions were filtered with a 70 µm cell strainer followed by subsequent removal of myelin, cell debris and erythrocytes. The cell suspension was then incubated with CD11b MicroBeads (Miltenyi Biotech) for 15 minutes at 4 °C followed by a separation in MS columns (Miltenyi Biotech) using a magnetic field on a MACS MultiStand and QuadroMACS (Miltenyi Biotech). Isolated microglia were labeled with unique oligo sample barcodes conjugated to anti- mouse CD45 antibodies (BD Mouse Immune Single-Cell Multiplexing Kit, #633793), pooled in equivalent amounts and loaded on a BD Rhapsody cartridge (BD Bioscience, #400000847) using a BD Rhapsody Express Single-Cell Analysis System. Cell counting was performed using Countess 3 FL automated cell counter (Invitrogen). Pooled samples were diluted using BD Sample Buffer to a final volume of 650µL. Approximately 60,000 cells in total were loaded on a BD Rhapsody cartridge. Cell and bead loading, cell lysis, recovery of mRNA-bound beads, reverse transcription, and exonuclease treatment were performed according to the manufacturer's instructions (BD Biosciences). Whole-transcriptome library preparation, based on the random-priming strategy, and index PCR steps were performed using the BD Rhapsody mRNA Whole Transcriptome Analysis and Sample Tag Library Preparation Protocol, as per the manufacturer's recommendations (BD Biosciences), optimized based on “Optimized workflow for single-cell transcriptomics on infectious diseases incl. COVID-19”, STAR Protoc., 2020. Quantification of the final libraries was performed using a Qubit Fluorometer with the Qubit dsDNA HS Kit (ThermoFisher), whereas the size distribution was assessed using the Agilent High-Sensitivity D5000 assay on a TapeStation 4200 system (Agilent Technologies). Paired-end sequencing (2∗75 cycles) was performed on a NovaSeq 6000 System (Illumina) using NovaSeq S1 Reagent Kit (200 Cycles) chemistry.