Ao pi viability dye

Patient MPNST samples were directly collected from the operating room in the Center for Clinical Research of the National Institutes of Health and immediately processed in a laminar flow hood. The tumor was minced into 1 mm³ cubes in tumor dissociation media {DMEM10, collagenase I (STEMCELL Technologies, Vancouver, Canada), dispase II (MilliporeSigma, St. Louis, MO), and DNase I (Invitrogen)}, and transferred to a gentleMACS C Tube (Miltenyl Biotec, Bergisch Gladbach, Germany) and dissociated on a gentle-MACS dissociator (Miltenyl Biotec) followed by shaking at 200 rpm at 37°C for 40 min. The dissociated tumor cells were pushed through a 40 mm cell strainer with a syringe plunge, and the cell strainer was rinsed twice with DMEM10. The cells were washed with PBS and counted using the AO/PI viability dye (Nexcelom, Lawrence, MA) in an automated cell counter, Cellometer Auto 2000 (Nexcelom) or LunaFL (Logos Biosystems, Annandale, VA). Ten thousand live cells per capture lane were subsequently captured by the Chromium Controller (10x Genomics, Pleasanton, CA), aiming to capture 6000 live cells per lane, using the v3 reagent for library preparation, following the manufacturer’s instructions. Sequencing was done on the Illumina sequencer NextSeq550, aiming for >50000 reads per cell.