GSM9151299: Aged; Mus musculus; RNA-Seq
Single cell RNA sequencing for endothelial cells Single cell analysis was performed on endothelial cells from eight mice per group (Young, Aged, Gpld1, TNAPi). Briefly, mice were sedated with ketamine followed by perfusion with 30 mL of ice-cold PBS. The entire brain was removed, then the hippocampus was sub-dissected. Single-cell suspensions were generated by enzyme-mediated (papain) and mechanical dissociation using Miltenyi Neural Dissociation Kits (P) (Miltenyi, 130-092-628) according to the manufacturer's instructions. The papain dissociation was done at 37°C for 10 minutes with three trituration steps. All other processing steps were performed at 4°C. Myelin was depleted from the suspensions using 22% Percoll solution (Cytiva, 17089101). Cell Fc receptors were blocked using a purified anti-mouse CD16/32 antibody (BD Biosciences, 553142) and then and then stained for 30 minutes at 4°C in the dark with CD45-PE (BD Biosciences, 553081), CD41-PE (BD Biosciences, 558040) and CD31-APC (BD Biosciences, 551262) and DAPI (Thermo Scientific, 62248 1:1000). Cells were sorted on a BD FACSAria Fusion with a 100 μm nozzle and with a flow rate of 1–2.5. Cells were first gated by forward and side scatter, then gated for doublets with area and height. Cells per sample that were DAPI-, CD45-,CD41-, CD31+ were collected for sequencing analysis. Isolated cells were given to the UCSF-CoLab Genomics Core for analysis with the 10x Genomics Chromium Single Cell Expression Solution 3′ kit. The Genomics Core prepared cells for 10x Genomics Chromium single-cell capture. 20,000 cells were loaded per sample. cDNA libraries were prepared according to the standard 10x Genomics protocols. The final library pool was sequenced on the NovaSeq 6000 or the NovaSeq X 10B system at the UCSF CAT Core. The raw base sequence calls were demultiplexed into sample-specific cDNA files with bcl2fastq/mkfastq and converted to count matrices using Cell Ranger 7.1 (10x Genomics).