GSM8712029: LAn3, replicate 3, scRNAseq; Mus musculus; RNA-Seq
The mouse liver tissues were surgically removed and first washed with Dulbecco's phosphate-buffered saline (DPBS) without calcium and magnesium to wash away blood and preservation medium, transferred into a petri dish and diced into small pieces (roughly 1 cm2 for resections and 0.25 cm2 for biopsies) using a scalpel. The liver tissue was dissociated for single-cell preparation using the mouse liver dissociation kit (Cat #130-105-807, Miltenyi Biotech, Auburn, CA) and gentleMACS Dissociator (Cat #130-093-235, Miltenyi Biotech, Auburn, CA.). Briefly, the tissue was distributed evenly into four gentleMacs C Tubes, then each tube received 5 ml of Liberase enzymatic digestion solution (0.2 Wünsch/ml) reconstituted in HepatoZYME media (without growth factors, which may influence the downstream scRNA-seq) containing DNAse I (2000 U/ml). The mixture was warmed to 37 °C for optimal enzymatic activity. The enzymatic digestion occurred in an incubating shaker at 37 °C and 200 RPM for 30 mins. The partially degraded extracellular tissue matrix in each C tube was mechanically dissociated by running two “B” cycles in the gentleMACS Dissociator. A 1:1 ratio of 20% FBS (Fetal Bovine Serum) to 80% DPBS was added to neutralize the enzymatic reaction and the cell suspensions were filtered through 70 um filters to remove large pieces of debris, gently mashing pieces through the filter. Cells dissociated from liver tissues were purified by centrifuging the cell suspensions at 50 x g, 4 °C for 5 mins to pelletize the cells; dissociated single cells were then stained for viability assessment using 0.05% trypan blue (Cat #T6146, Millipore Sigma, St. Louis, MA) in PBS on a Cellometer AutoT4 cell counter (Nexcelom Bioscience, Waltham, MA). Library constructed according to 10x Genomics single cell 3' v2 protocol