title: GSM8639092: young3; Mus musculus; ncRNA-Seq
library_name: GSM8639092
library_construction_protocol: Microglia from young and aged mice (3 and 21 months, C57BL/6, males) were isolated via FACS-sorting following the below described protocol. In brief, mice were anaesthetized with Avertin and perfused with 20 mL ice cold DPBS. Brains were dissected, hemispheres separated, and olfactory bulb and cerebellum removed. Single-cell solutions were created for each hemisphere via mincing and douncing the tissue, the solution was filtered (70µm cell strainers, Falcon 352350) and finally centrifuged (400 x g, 10 mins, 4°C). After resuspension in MACS buffer and addition of myelin removal beads (Miltenyi Biotech, 130-096-433), solutions for each hemisphere were loaded on LD columns (Miltenyi Biotech, 130-042-901) to remove myelin. Columns were washed twice with MACS buffer. Cells were pelleted via centrifugation (400 x g, 5 mins, 4°C) and resuspended in FACS buffer. FC blocking antibody was added and incubated for 5 mins. Primary antibodies (CD11b-FITC (Biolegend 101206); CD-45-BUV396 (Biolegend 50-162-785) and CD206-APC (Biolegend 141707)) were added and incubated for 30 mins on ice. After another centrifugation, samples were resuspended in 0.5 mL FACS buffer and Sytox Blue (Thermo, S34857) was added for live cell labelling. Cells were sorted on a MA900 Multi-Application Cell Sorter (Sony Biotechnology) and selected by gating for live single cells (FSC-A/SSC-A). Further, cells were gated for CD11b+; CD45low; CD206- and sorted directly into 1.5mL tubes containing 50µl FACS buffer. As CD11b+/CD45+ brain cells are mainly infiltrating macrophages (PMID: 32381088) we exclude these macrophages with our sorting strategy. FACS-isolated CD11b+/CD45low/CD206- brain myeloid cells are referred to as microglia in this manuscript. Between 10,000 and 40,000 cells per sample were collected. For aged mouse sample 4, two mice were pooled together to reach the threshold of minimal 10,000 cells as input for RNA isolation. After collection was completed 1 mL QIAzol was added instantly. After vortexing, samples were stored at -80°C until RNA isolation. Since cell sorting provides little input material for RNA isolation, the standard miRNeasy microKit (QIAGEN, cat. no. 217084) was used with standard protocol for elution of separated fractions for RNAs above and below 200 nt, to concentrate the miRNA input for library preparation. Library preparation of microglia miRNAs was optimized and performed as described below, except for the RT-primer input, which was altered to a 1:5 dilution and amplification PCR was 25 cycles long. The MGIEasy Small RNA Library Prep Kit (Item 940-000196-00) was used for library preparation on the high-throughput MGI SP-960 sample prep system according to the manufacturer's protocol. In principle this library preparation method works by ligating 3'- and 5'-adapters to all RNAs in each sample. During reverse transcription (RT), specific RT primers that bind to the adapters are used to generate cDNA and introduce sample specific barcodes. Amplification of this cDNA is performed via a 21-cycled PCR. Size-selection of this PCR product is performed via magnetic beads (AMPure Beads XP, Beckman Coulter). To focus on the small RNAs a size of around 110 bp was selected, this was checked using an Agilent DNA 1000 Kit (Agilent Technologies). The concentration of each sample was measured by a QuBit 1x dsDNA High Sensitivity Assay (Thermo Fisher Scientific). All samples were pooled in one library after concentration measurement in an equimolar fashion to reach a concentration of 4.56 ng µl-1 for each sample in each pooled library. After circularization the pooled libraries were sent for sequencing.
identifiers.namespace: GEO
identifiers.id: GSM8639092_r1