Homo sapiens
Protocols: Primary tumor cells at passage 1 were dissociated through trypsin/EDTA (Lonza cat. no. BE17-161F), centrifuged at 300 x g for 5 min, the supernatant was removed and the cell pellet was washed twice with pre-warmed Dulbecco-s Phosphate Buffered Saline (D-PBS 1X) and incubated with 0.25% trypsin/EDTA solution at 37 °C until cells are detached from the bottom of the flask. Cells were collected in D-PBS, pelleted at 300 x g for 5 minutes at RT, suspended in 500l of pre-warmed stem media (MEBM) and counted both with TC-20 automated cell counter (Bio-Rad) and counting chamber (Biosigma-Fast Read 102).   The cells were centrifuged at 300 x g for 5 minutes, and the pellet was resuspended in pre warmed serum-free MEBM supplemented with 100U/ml penicillin, 100ug/ml streptomycin, 2mML-glutamine, 5ug/ml insulin, 0.5ug/ml hydrocortisone, 1U/ml heparin,2% B27, 20ug/ml epidermal growth factor, 20ug/ml fibroblast growth factor and diluted by serial limiting dilution at the density of 1 cell for well  in the media supplemented with 12.5% of ascitic fluid for plating into  low cell adhesion 96 well plates (Sumitomo Bakelite cat. no.  MS-9096V 96 well) with a final volume of 200 l for well. sMOCS at day 8-11 was moved from  96-well V-bottom ultra-low attachment plates to 48-well ultra-low attachment plates (Corning), and incubated with 400l of pre-warmed 0.25% trypsin/EDTA solution at 37 °C  for 20-30 minutes with gently pipetting for 20 times every 8 minutes and visualized by microscope for the stadium of disaggregation. To avoid loose of cells the tip was always rinsed with medium before pipetting. Cells were harvested, washed once in MEBM plus ascitic fluid and centrifuged at 300 x g for 5 min. Chromium Next GEM Single Cell 3′ GEM, Library & Gel Bead Kit v2 Chromium Next GEM Single Cell 3′ GEM, Library & Gel Bead Kit v2