title: GSM8714227: GEMM KPT UPS; Mus musculus; RNA-Seq
library_construction_protocol: For isolation of tumor cells from KPT mice and PDXs, mice were euthanized at the indicated time points. Dissected tumors were dissociated with a mixture of Dispase II, Collagenase Type IV, and DNase I for 1 hr. The dissociated cells were filtered using a 100 µm filter and spun at 1000 rpm for 10 min at 4 °C. The supernatant was removed by aspiration and red blood cell lysis was performed using BD Pharm Lyse for 1 minute on ice. Cells were then washed with sterile media containing 2% heat inactivated FBS, passed through a 40 µm filter, and pelleted at 300 g for 5 min at 4 °C. The supernatant was removed and cells were resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (2% heat-inactivated FBS in PBS) and counted for use in FACS. Single cell suspensions from UPS tumors were prepared and stained as above. Live sorted cells were collected by flow cytometry, washed once with PBS containing 2% heat inactivated FBS (HI-FBS) and resuspended to a final concentration of 700–1,300 cells per μl of SMEM + 2% HI-FBS. The viability of cells in all experiments was confirmed to be above 80% by 0.2% (w/v) Trypan Blue staining (Invitrogen, Countess II) and processed by droplet based scRNA-seq using the 10X genomics Chromium Single Cell 3' Library & Gel bead Kit >=V3 according to manufacturer's protocol. Following reverse transcription and cell barcoding in droplets, emulsions were broken, and cDNA purified using Dynabeads MyOne SILANE followed by PCR amplification as per manufacturer's instructions. Between 10,000 to 30,000 cells were targeted for each droplet formulation lane using Chromium. Samples were multiplexed using the TotalSeq B cell hashing protocol (Biolegend). Final libraries were sequenced on Illumina NovaSeq S4 platform (R1 – 28 cycles, i7 – 8 cycles, R2 – 90 cycles). 