title: single-cell RNA-seq; Homo sapiens; tumor of the endometrium: Sample 36186L_RNA
design: Tumor specimens were minced using two razor blades and digested overnight in 20-30 mL DMEM/F12 + 5% FBS, 15mM HEPES (Gibco), 1x Glutamax (Gibco), 1x Collagenase/Hyaluronidase (Stem Cell Technologies), 1% Penicillin/Streptomycin (Corning), and 0.48 µg/mL Hydrocortisone (Stem Cell Technologies) on a stir plate at 37°C and 180 rpm. For ovarian tumors, Gentle Collagenase/Hyaluronidase (Stem Cell Technologies) was used instead of Collagenase/Hyaluronidase. After digestion, tumor cells were washed twice with cold PBS + 2% FBS and 10mM HEPES (PBS-HF) and centrifuged at 1200 rpm for 5 min at room temperature.  To remove red blood cells, the cell pellet was treated with 4 or 8 mL cold Ammonium Chloride Solution (Stem Cell Technologies) with 1 or 2 mL PBS-HF, respectively, for 1 minute, then centrifuged at 1200 rpm for 5 min. The amount of Ammonium Chloride Solution added was based on the size of the cell pellet and visual assessment of pink or red color present in the pellet. This step was repeated a second time if the pellet still exhibited a pink or red color after initial treatment. To further dissociate the cells, pellets were resuspended in 1-2 mL 0.05% Trypsin-EDTA (Gibco) and the suspension was gently pipetted up and down for 1 min. After 1 min, trypsin was inactivated by adding 10mL PBS-HF solution. The suspension was then centrifuged at 1200rpm for 5 min. If cell suspensions were clumpy, cells were resuspended with 1-2 mL Dispase (Stem Cell Technologies) and 200 µL 1mg/mL DNase I (Stem Cell Technologies) for 1 min, then inactivated with 10 mL PBS-HF. If the Dispase step was not necessary, cells were treated with DNase I during the trypsinization step. Cells were again centrifuged at 1200 rpm for 5 min, then washed in 10 mL PBS-HF and filtered through a 100µm cell strainer. A final centrifugation step was done at 1200 rpm for 5 min. The cell pellet was resuspended in DMEM/F12 + 5% FBS using a volume based on the final pellet size and filtered using a 40µm cell strainer. Single-cell suspension concentration and cell viability was measured by adding 10 µL 0.4% Trypan Blue to 10 µL cell suspension and measuring with the Countess II Automated Cell Counter (Thermo Fisher, AMQAX1000). We aimed for cell viability above 60% for the cells to be used for single-cell sequencing. Cell viability varied between 64% and 94% across all samples, with the majority of tumor suspensions being over 70% viable.  To continue with scRNA-seq, the cell suspension was diluted to 1200 cells/µL and 10,000 cells were used in library preparation using the 10x Genomics Chromium Single Cell 3' GEM Kit v3 (PN-1000077) following the manufacturer's protocol.  Libraries were sequenced on an Illumina NextSeq500 in the pair-end format using 10x Genomics recommended sequencing parameters. Pooled samples were demultiplexed with cellranger mkfastq.