title: GSM9557119: KPC PDAC tumor microenvironment, sample Z204; Mus musculus; RNA-Seq
library_construction_protocol: Single-cell suspensions were prepared from dissociated whole pancreatic tumors harvested from the KPC mouse model of pancreatic ductal adenocarcinoma (PDAC). Briefly, individual tumors were minced into small fragments and digested in RPMI 1640 medium supplemented with 1 mg/mL collagenase IV, 0.5 mg/mL Dispase II, and 40 U/mL DNase I at 37°C for 20-30 minutes with gentle agitation. The digested tissue was then passed through a 70-μm cell strainer and washed with PBS containing 0.04% BSA. Red blood cells were lysed using ACK lysis buffer. Cell viability and concentration were assessed using trypan blue staining on an automated cell counter. To remove dead cells and debris, the suspension was processed through a Miltenyi Biotec Dead Cell Removal Kit according to the manufacturer's instructions. Finally, the live single-cell suspension was resuspended in PBS with 0.04% BSA for loading onto the 10x Genomics Chromium Controller to generate gel bead-in-emulsions (GEMs) for subsequent library construction. Single-cell RNA-seq libraries were constructed in accordance with the manufacturer's instructions. In brief, the prepared live single-cell suspension was combined with master mix, partitioning oil and gel beads containing barcoded oligonucleotides. This mixture was then loaded onto a Chromium chip to generate gel bead-in-emulsions (GEMs). Within each GEM, the cells were lysed and the poly-adenylated mRNA was reverse transcribed into barcoded cDNA. This process incorporated a unique molecular identifier (UMI), a cell barcode and an Illumina R1 sequence. Following GEM breakage, the barcoded cDNA from all GEMs was pooled and purified using DynaBeads MyOne SILANE beads. This was then amplified by PCR to generate sufficient material. The amplified cDNA was then fragmented enzymatically, with Illumina adapter sequences and a sample index added during the end repair, A-tailing, adaptor ligation and PCR amplification steps. Final library fragments optimised for sequencing were selected to enrich for constructs of an appropriate length (200–700 bp). The quality of the library was assessed based on fragment size distribution and quantified prior to pooling and sequencing.