GSM7501731: B16F10 metastatic lung; Mus musculus; RNA-Seq
For sorting, tissues were minced on ice and digested for 1 h at 37°C using 1 mg ml-1 collagenase Type I (Gibco/Thermo Fisher Scientific), 0.1% BSA and 7.5 µl ml-1 of DNAseI (Roche, Basel, Switzerland) in DPBS (Gibco/Thermo Fisher Scientific). Thereafter, samples were vortexed and passed through a 40 µm Nylon cell strainer (Corning Life Sciences or Thermo Fisher Scientific), centrifuged at 1200 rpm for 7 min, resuspended in red blood cell (RBC) lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA) for 2 min and washed using 0.1% BSA, 2 mm EDTA in PBS (FACS buffer).  Cells were stained using anti-Pecam1-PE-conjugated antibody in FACS buffer for 30 min, washed twice with FACS buffer and sorted using BD InfluxTM (BD Biosciences, Franklin Lakes, NJ, USA). Anti-Pecam1-PE positive cells were used for 10x library preparation using the Chromium Single Cell 3´ Reagent Kits v2 (10x Genomics; Pleasantom; CA, USA). The cell recovery aim for each library was 8000 cells. Libraries were sequenced using NovaSeq 6000 system (Illumina, San Diego, CA, USA) using S4 flow cell with read length of 28+8+89. Sequencing was performed by the Sequencing laboratory of Institute for Molecular Medicine Finland FIMM Technology Centre, University of Helsinki.