GSM5145935: MG_scRNA-seq; Mus musculus; RNA-Seq
Mice were anesthetized with Avertin (250 mg/kg of body weight, i.p.) and transcardially perfused with 25 mL of cold 0.1 M PBS, pH 7.4. For Cx3cr1GFP and Trem2ko mice used for FACS, flow cytometry and single cell RNAseq the infusion sites in both hemispheres were excised for cellular isolation as described below. For WT mice, one hemisphere was drop fixed in 4% phosphate-buffered paraformaldehyde at 4 °C for 48 h before storage in 30% sucrose. The other hemisphere was used for cellular isolation as described below. Fixed hemibrains were mounted in O.C.T. and cut in the coronal plane at 30 µm sections using a frozen cryotome (Thermo Scientific) and stored in glycol-based cryoprotectant at -20 °C until histological staining. Prior to staining, sections containing the infusion site were selected under a dissection microscope (Olympus). In a separate cohort of APP/E3 and APP/E4 mice (14 mo.) CSF was collected prior to perfusion. Once anesthetized a small incision was made exposing the foramen magnum, a 28g 1/2" needle was used to puncture a hole at the base of the skull and a capillary tube was applied for CSF withdraw. scRNA libraries were generated with Chromium single cell 3' chip kit v3 for scRNA-seq (10x Genomics) according to the manufacturer's instructions and then the quality assessed using Agilent Bioanalyzer 2100. Sequencing was performed using NovaSeq (Illumina) by MedGenome Inc. with a custom sequencing setting (28bp for Read1 and 91bp for Read2) to obtain an average sequencing depth of 45K reads/cell.