GSM8493768: Tumor microenvironment Lysm WT; Mus musculus; RNA-Seq
Microdissected liver metastases were chopped with scalpels. Subsequent enzymatic digestion was performed with LiberaseTM (Roche, 5401119001), 10mg/ml DNase I (Roche, 10104159001) in DMEM (Invitrogen, 41966-029) for 90 min at 37 °C, in a shaking water bath. Digested tissue fragments were quenched, filtered through 70μm cell strainer and subsequently, washed with FACS Buffer (PBS with 1% BSA (Roche, 10735086001), 2mM EDTA (Sigma, E5134-500G), 0.05% NaN3 (Sigma, S2002-25G)).  Dead cells were stained with either LIVE/DEAD™ Fixable Dead Cell Stain Kit Yellow, Aqua (ThermoFisher, L34959 & L34957) or Zombie Violet™ Fixable Viability Kit (BioLegend, 423113) or DAPI (Sigma Aldrich, D9542). Samples were Fc-blocked with anti-CD16/32, and then cells were stained for the extracellular markers for 25 min at 4°C. 10x Genomics 3-prime v3.0 (TME and CAFs) and 5-prime v1.0 (T cells)