GSM4971322: 161_Aged_High_scRNA-seq; Mus musculus; RNA-Seq
After collection, fresh whole brains were dissociated using the Adult Brain Dissociation kit (Miltenyi Biotec, Germany), for 30 min at 37oC. After filtration (70µm), debris and red blood cells were removed according to the kit protocol, and the obtained pellet was depleted of myelin and dead cells, using the Myelin Removal Beads v2 (Miltenyi Biotec, Germany), and the Dead Cell Removal Kit (Miltenyi Biotec, Germany), sequentially. Final pellets were eluted in 100µL of PBS with 4% BSA. Single-cell 3' cDNA libraries were prepared at the DNA Services laboratory of the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign. Single-cell 3' cDNA libraries were prepared at the DNA Services laboratory of the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign. The four single-cell suspensions with an average viability of 75.5 ± 3.1 %, and concentration of 753 ± 121.3 cells/µL by AO/PI staining on the Nexcelom K2 (Nexcelom Bioscience, Lawrence, MA) were converted into individually barcoded cDNA libraries with the Single-Cell 3' Chromium kit version 3 from 10X Genomics (Pleasanton, CA) following the manufacturer's protocols. The target number of cells per library was 6000. On average 14.5 ± 2.2 µL of the cell suspensions were used for the preparation of the libraries. The 10X Chromium instrument separates thousands of single cells into Gel Bead Emulsions (GEMs) that add a barcode to the mRNA from each individual cell. Following ds-cDNA synthesis, individually-barcoded libraries compatible with the Illumina chemistry were constructed. The final libraries were quantitated on Qubit and the average size determined on the AATI Fragment Analyzer (Advanced Analytics, Ames, IA). Libraries were pooled evenly, and the final pool was diluted to 5nM concentration and further quantitated by qPCR on a Bio-Rad CFX Connect Real-Time System (Bio-Rad Laboratories, Inc. CA). The final library pool was sequenced on one lane of an Illumina NovaSeq 6000 S4 flowcell as paired-reads with 28 cycles for read 1, 8 cycles for the index read, and 150 cycles for read 2.