title: GSM9168138: Aorta, Tagln Cre -, Prdm16 fl/fl, Baseline; Mus musculus; RNA-Seq
library_name: GSM9168138
library_construction_protocol: scRNAseq was performed using the 10x Flex Platform with the Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit (1000414). Aortae were dissected from the root to the ileac bifurcation, washed twice in cold RPMI. The tissue was then sliced in 1 mm rings on a chilled glass slide and fixed in 1 mL fixation buffer per 25 mg tissue for 16h at 4°C without agitation. The fixation buffer consisted of 4% PFA combined with 10x concentrated fixation/permeabilization buffer). The following day, tissue pieces were centrifuged at 850 x g for 5 min and washed with 2 mL cold PBS. Samples were then resuspended in 1 mL quenching buffer with 0.1 volume of Enhancer, and adjusted to a final glycerol concentration of 10% before being stored at -80°C. For enzymatic digestion, aortae were processed individually in 2 mL pre-warmed RPMI 1640 containing 1 mg/mL Liberase TM (Roche 05401020001) using a Miltenyi GentleMACS at 37°C with the following program: (spin 50 rpm for 20 min, [spin 1000 rpm for 30s, spin -1000 rpm for 30s (2x)]). Digested tissue suspensions were pooled and filtered through a 30 µm strainer. The strainer and collection tube were rinsed with PBS to maximize cell recovery. Cells were centrifuged at 1200 × g for 10 minutes and resuspended in 250 µL quenching buffer Probe hybridization and cell capture were performed using the 10x Chromium Fixed RNA Kit, Mouse Transcriptome kit (1000495) according to the manufacturer's instructions except for an increase in centrifugation speed (1200 x g for 10 min).