title: GSM8790406: KPC tumor, control treatment, biological replicate 3; Mus musculus; RNA-Seq
library_construction_protocol: Single-cell suspensions were prepared using the Mouse Tumor Dissociation Kit (Miltenyi Biotec) from mouse pancreatic tumors. Cells were frozen in Bambanker (NIPPON Genetics) and stored in -80°C until sequenced. Once ready, cells were quickly thawed in a 37°C bath and transferred into a 15 ml conical tube with 4 ml 1% BSA in PBS (Sigma-Aldrich). The resuspended cells were filtered using a MACS™ 30 µm filter (Miltenyi Biotec) and centrifuged at 300 RCF for 5 minutes at 6°C. Supernatant was removed from the cells with care taken not to disturb the pellet, and the cells were then resuspended in 1% BSA in PBS, with volumes adjusted depending on the pellet size. Cells were quantified using a fluorescent cell counter with 1:1 trypan blue (Bio-Rad) and 1:1 AO/PI viability assay (DeNovix). For the KPC collagen gel characterization studies and tumors from Col1a1CreERT2 MyD88fl/fl mice and control mice comparison studies, cells from each individual sample were labeled separately with TotalSeq™-B hashtag antibodies (BioLegend). Target input cell range was 700-1200 cells/µl with a target cell recovery of 10,000 cells. Library preparation was performed using Chromium Next GEM Single Cell 3' Reagent Kits v3.1 and sequenced at the Stanford Functional Genomics Facility on the Illumina NovaSeq 6000.  Target input cell range was 1300-1600 cells/µl with a target cell recovery of 20,000 cells. Library preparation was performed using Chromium GEM-X Single Cell 5' Reagent Kits v3 (10x Genomics), per manufacturer's protocol. Library conversion was then done with Singular Genomics Library Compatibility Kit (Singular Genomics), and libraries were sequenced on the Singular Genomics G4 sequencer, using 100 cycle F3 flow cells (Singular Genomics), with two lanes per library at 28/90 paired end.