title: GSM9604174: isolated living cells from aged mouse brain after FL-RSV treatment_2; Mus musculus; RNA-Seq
library_construction_protocol: After euthanasia, mice were transcardially perfused with ice-cold PBS, and brains were immediately collected into ice-cold PBS. For the scRNA-seq experiment, brains were obtained from young and aged mice, with n = 3-4 per group. The brains were then washed in ice-cold PBS and cut into ~1 mm pieces. Single-cell suspensions for scRNA-seq were generated by adapting the authors' brain flow-cytometry dissociation protocol. In the base protocol, the olfactory bulb, cerebellum, and brainstem were removed, and meningeal vessels were stripped by rolling the brain on Whatman paper. The tissue was finely chopped, and enzymatic digestion was performed using 50 μL collagenase, 2 μL elastase, 2 μL dispase I, and 3 μL hyaluronidase for 45 min at 37°C in a rotating incubator. Following digestion, the tissue was mechanically dissociated by pipetting with 10 mL and 5 mL pipettes, filtered through 100 μm and subsequently 30 μm nylon mesh, and centrifuged. For cleanup, Debris Removal Solution (Miltenyi Biotec) was used. In the flow-cytometry protocol, cells were resuspended in 3.1 mL PBS, 0.9 mL Debris Removal Solution was added, and the mixture was inverted five times to mix. The suspension was then slowly overlaid with 4 mL PBS and centrifuged at 3000 × g for 10 min. After centrifugation, the top buffer layer and the white myelin–debris interphase were removed, and the pelleted cells were washed in PBS and centrifuged again at 1000 × g for 10 min at 4°C. The scRNA-seq section confirms that digestion and purification using Debris Removal Solution were performed, although every enzyme quantity and centrifugation parameter was not explicitly restated; therefore, these values were inferred from the referenced flow-cytometry protocol that was adapted. After cleanup for scRNA-seq, the cell pellet was resuspended in PBS + 0.1% BSA and stained with SYTOX Green to identify live cells. The suspension was then subjected to low-pressure FACS using the WOLF Cell Sorter (NanoCellect) to remove debris and dead cells and to enrich for viable cells. Cells were maintained on ice pending sequencing. After sorting, cells were centrifuged at 2200 rpm for 10 min at 4°C and finally resuspended in 30–50 μL of 0.04% BSA, with the volume adjusted according to the number of recovered cells. Following cell sorting, the recovered viable cells were processed using a droplet-based single-cell RNA sequencing workflow on the Chromium platform (10x Genomics). In this system, individual cells were partitioned into nanoliter-scale droplets together with barcoded gel beads to generate gel bead-in-emulsion (GEM) partitions. Within each GEM, cell lysis occurred and reverse transcription was carried out, allowing newly synthesized cDNA molecules to be labeled with both a unique molecular identifier (UMI) and a cell-specific barcode. This barcoding strategy enabled transcripts originating from individual cells to be distinguished during downstream analysis. After reverse transcription, the GEMs were broken and the pooled barcoded cDNA was recovered and purified. The resulting cDNA was then amplified through PCR to generate sufficient material for library preparation. Amplified cDNA products were subsequently processed through a series of enzymatic steps to generate sequencing-compatible fragments. During this stage, cDNA molecules were fragmented and end-repaired to produce fragments of an appropriate size distribution for sequencing. Fragmented DNA was then subjected to adaptor ligation so that sequencing adaptors required for Illumina sequencing platforms were incorporated. Following adaptor ligation, additional purification and size-selection steps were performed to remove small fragments, adaptor dimers, and other reaction by-products. The adaptor-ligated DNA fragments were then amplified through sample index PCR to introduce sample-specific index sequences and to produce the final sequencing-ready library. These index sequences enabled multiplexing of multiple samples within the same sequencing run while maintaining the ability to demultiplex reads during data processing. After library amplification, final cleanup and size-selection steps were performed to ensure a consistent fragment size distribution and to remove residual primers or adaptor fragments. Library quality and fragment size distribution were then assessed using capillary electrophoresis-based quality control. Libraries were quantified, pooled according to molar concentration, and prepared for sequencing.