Sample 3_p
Before euthanizing the mice, tumor dissociation solution was prepared by adding 4mL of 10X collagenase A in 26 mL of DMEM F12, 10mL of BSA fraction V and 0.5mg/mL DNase and was kept on ice. Primary tumor was harvested and washed with cold 1XPBS. Tumor section (~3 cm3 ) was placed in a 10cm dish containing 5mL of dissociation solution. Tumor tissue was minced with a scalpel into small pieces (~1mm3 ) and transferred to 10mL tubes. Another 5mL of dissociation solution was added and the tubes were placed in 370C incubator for 20 mins rotating slowly in a Mini Tube Rotator (Fisher Scientific). The tissue suspension was passed through 70μm cell strainer for 53 twice. The left-over unfiltered tissue was grinded with a 3ml syringe plunger. Cell pellet was collected by spinning at 300g for 5mins at 40C, resuspended and incubated in 1mL RBC solution for 10min at room temperature. After centrifugation, cell pellet was resuspended in 2mL of PBS-based washing buffer containing 0.5% BSA. Cell suspension was incubated with 7.5μg/mL E-cadherin (CD34, eBioscience) antibody for 10 min to deplete tumor cells by using Dynabeads® Sheep Anti-Rat antibody (ThermoFisher Scientific). The remaining cells were used to enrich CD31-positive endothelial cells by using 7.5μg/mL anti-CD31 antibody (eBioscience) and the EasySep™ PE Positive Selection Kit II (Stemcell Technologies). The obtained cells were resuspended in 1mL PBS + 0.04% BSA and were used for single cell sequencing by 10X genomics (CA, USA). Libraries for single-cell RNA sequencing were prepared according to 10X Genomics procedures (Pleasanton, CA). Briefly, Samples with >95% viability were selected and resuspended with the appropriate volume of 1X PBS + 0.1% BSA to reach a concentration of 1,000 cells/mL.  The cell suspension was then used to generate the gel bead-in emulsions, reverse transcription, cDNA amplification, library preparation, and sample indexing following the latest available manufacturer's protocol, using the 10X Chromium Controller (PN-1000202), the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3 (PN-1000075), the Chromium Next GEM Chip B Single Cell Kit (PN-1000073) and the Single Index Chromium i7 Multiplex Kit, (PN-120262).