title: GSM8545170: APOE_SFD, scRNA_seq; Mus musculus; RNA-Seq
library_construction_protocol: All animals were anaesthetized by isoflurane inhalation (3%) and euthanized by exsanguination. Aortas from 4 mice of each group were harvested and dissected into small pieces. Tissues were digested in a 1.5 mL digestive solution containing 1.5 mg/mL Collagenase A (Roche, 10103578001) and 0.5 mg/mL Elastase (Sigma-Aldrich, E1250) dissolved in DMEM (Gibco) with 10% FBS (Noverse, NFBS2500A) at 37°C for 3 h. Suspension was slowly pipetted up and down using sterile pipette tips every half-hour. After digestion, the solution was transferred into flow tubes, and 3 mL of flow buffer (i.e., PBS with 10% FBS) was added to terminate the digestion. After centrifugating at 500 g for 5 min at 4°C, the supernatant was discarded, and the pellet was washed with 1 mL flow buffer followed by centrifugation as described above. Cell pellets were then resuspended in 150 μL flow buffer and incubated with fluorochrome-coupled antibodies against CD31 (Invitrogen, 11-0311-82) and CD45 (Invitrogen, 12-0451-82) at 1:100 for 30 mins on ice in the dark. After incubation, cells were washed with 1 mL flow buffer and resuspended in 300 μL PBS. 4,6-Diamidino-2-phenylindole (DAPI) (1:1000) was added to mark dead cells before sorting. The resulting cell suspension was filtered using 40 μm cell strainers. The CD45− and CD31+ living cells were then acquired using a flow sorter (BD FACS ORP ARIA II). Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.