GSM8000488: PLV Biological replicate 1; Mus musculus; RNA-Seq
To obtain single cell suspensions from popliteal lymphatic (PLVs), adult WT1xAi9 mice were anesthetized with K/X (0.2 mL/20g) and administered 4% Evans blue dye footpad injections. Popliteal vessels were then exposed, and we used a previously described method to generate single cell suspensions of the vessels. Briefly, minced vessels were incubated for 30 minutes at 37ºC with agitation in an enzymatic cocktail consisting of 1 mg/ml collagenase I (Worthington, LS004196), 1 mg/ml Dispase II (Gibco, 17105041), DNAse (Milipore Sigma, 04716728001) and Liberase (Milipore Sigma, 05401020001). The resulting cell suspension was then filtered through a 70 µm nylon filter (Fisher Scientific, 22363548) and digestion enzymes were inactivated by adding FBS. In total, we performed this protocol on two sets of two mice each. Our scRNA-seq library was made using the Next GEM Single Cell 3' Reagent Kit (10X Genomics) following the manufacturer's instructions