GSM8195546: CMG_rep4; Mus musculus; RNA-Seq
MBM samples preparation This protocol was performed in cold to prevent microglia ex vivo activation. Brains were sliced in 1 mm thick cuts and placed under the stereomicroscope, MBM were dissected manually and placed in ice-cold HBSS containing 15 mM HEPES and 0,5% Glucose. Control tissue or MBM were mechanically disrupted with 5X slow strokes with a Dounce tissue grinder (Wheaton, 357542) on ice. Samples were centrifuged at 350g during 5 min at 4°C and later at 8.000 g during 30 sec. Samples were subjected to RBC lysis buffer (Biolegend, 420301) for 3 min at RT and then spined again as described. Samples were subsequently subjected to flow cytometry, FACS or scRNAseq library preparation. FACS and flow cytometry: Prior to antibody staining, samples were blocked with Fc-block CD16/CD32 (Biolegend, 101320, 1:50) in FACS Buffer sterile filtered (1% FCS, 2mM EDTA, 25mM HEPES in PBS) for 10 min on ice and darkness to block nonspecific binding. For cell surface staining, cells were resuspended in 26 the appropriate antibody cocktail and incubated for 30 min on ice protected from light. Samples were centrifuged and washed with FACS buffer. Viability was assessed by staining with DAPI (1 μg/mL). Information and dilution for antibodies used for flow cytometry are listed in supplementary table 10. Immune cell profiling by flow cytometry was carried out by analyzing 50.000 live singlets in each sample with a FACS Aria III instrument and BD FACSDiva (BD Bioscience). After MBM sample preparation as described previously, samples were resuspended in FACS Buffer and subjected to surface antibody staining for TMEM119 (ab210405) and CD49D (Biolegend, 103627) and DAPI for 30 minutes on ice and darkness and subjected to FACS. Cells were collected in extraction buffer from the Arcturus PicoPure RNA Isolation Kit (Thermo Fisher). Then, following manufacturer's instructions RNA was eluted in TE buffer and 1 μL subjected to Bioanalyzer High Sensitivity RNA chip (Agilent) for quality testing. Four samples containing isolated cells from MBM and control brains from 3 independent mice (males and females) Stand-specific (reverse) libraries were prepared with cDNA converted using the SMARTer universal low input RNA synthesis kit. Sequencing was performed in single-end configuration with a read length of 150 bp.