GSM8198243: MASH+RvD1 liver scRNA rep1; Mus musculus; RNA-Seq
Mouse liver non-parenchymal cells (NPC) for scRNA-seq were isolated. Briefly, the liver was perfused in situ with HBSS buffer (without calcium & magnesium) containing 0.2 mg/mL EDTA, followed by sequential perfusion with HBSS buffer (with calcium & magnesium) containing 0.4 mg/mL pronase (Sigma, P5147) and 0.2% collagenase type II (Worthington, LS004196). The liver was minced and further digested with HBSS buffer (with calcium & magnesium) containing 0.2% collagenase type II, 0.4 mg/mL pronase and 0.1mg/mL DNase I (Roche, R104159001) in 37 C incubator with shaking for 30 min. Digestion was then stopped with DMEM containing 10% serum. The resulting liver cell suspension was passed through 100 mm nylon cell strainer and centrifuged at 50 g for 3 min three times to remove hepatocytes. This NPC suspension was centrifuged at 800 g for 5 min. The NPC cell pellet was subjected to red blood cell lysis buffer for 2 min. Upon the removal of red blood cells, the NPC cells were resuspended in HBSS, and subjected to density gradient centrifugation using 20% Optiprep (Axis Shield, # 1114542) to remove dead cells. Cell viability was confirmed by trypan blue exclusion. scRNA-seq libraries were prepared using a Chromium Single Cell 3' Reagent kit (10x Genomics) according to the manufacturer's instructions.