Systematic Single-Cell Dissection of Cell Cycle and TGFβ-Induced State Transitions Underlying Gemcitabine Resistance in 3D Pancreatic Tumor Tissue
Systematic Single-Cell Dissection of Cell Cycle and TGFβ-Induced State Transitions Underlying Gemcitabine Resistance in 3D Pancreatic Tumor Tissue
Title: 3D culture of PANC-1 pancreatic cancer cells on SISmuc scaffolds Description: PANC-1 human pancreatic ductal adenocarcinoma cells were cultured in 3D on decellularized porcine small intestinal submucosa (SISmuc) scaffolds as described in Dandekar et al. (2023). Scaffolds were seeded with PANC-1 cells and maintained under standard conditions (37 °C, 5 % CO₂, DMEM supplemented with 10 % FBS and 1 % penicillin–streptomycin). At experimental endpoints, cells were recovered from 3D scaffolds using collagenase digestion and washed in PBS prior to single-cell suspension preparation.  Parameters:  Organism: Homo sapiens  Cell line: PANC-1  Culture type: 3D SISmuc (decellularized porcine jejunum)  Medium: DMEM + 10 % FBS  Temperature: 37 °C  CO₂: 5 % Title: Recovery of cells from SISmuc 3D scaffolds Description: Following treatment, scaffolds were washed in PBS and digested with 1 mg/mL collagenase IV (37 °C, 30 min). Cells were passed through a 40 µm filter, centrifuged (300 × g, 5 min), resuspended in PBS + 0.04 % BSA, and counted using a hemocytometer. Viability > 85 % was required for single-cell capture. Title: Maintenance and expansion of PANC-1 cells Description: PANC-1 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 10 % fetal bovine serum (FBS) and 1 % penicillin–streptomycin under standard incubator conditions (37 °C, 5 % CO₂). Cells were subcultured at 70–80 % confluence. Title: Gemcitabine and TGF-β1 treatments in 3D PANC-1 cultures Description: 3D PANC-1 cultures were treated with gemcitabine (10 µM, 24 h), recombinant human TGF-β1 (10 ng/mL, 48 h), or the combination thereof. Control scaffolds were treated with vehicle (DMSO) under identical conditions. After treatment, cells were processed immediately for single-cell capture. Title: Conversion of 10x Cell Ranger outputs for downstream FAIR data archiving Description: Output matrices and metadata were converted to Annotare-compatible tab-delimited formats (annotare_metadata.tsv) and uploaded via EBI FTP. MD5 checksum verification ensured data integrity. Title: Single-cell preparation, CellPlex labelling and GEM generation for 10x Genomics Chromium Description: Single-cell suspensions were prepared by enzymatic dissociation and filtration through 40 µm strainers; cell viability exceeded 85 %. Each of the eight biological samples was labelled individually with a 10x Genomics CellPlex cell multiplexing oligo (CMO), washed to remove unbound CMO, counted, and combined in equal proportions into a single pooled suspension. Approximately 10,000 viable cells from the pool were loaded into a 10x Genomics Chromium Controller (Single Cell 3′ v3 chemistry). Cells were lysed within Gel Bead-in-Emulsion (GEM) droplets, and both mRNA and CMO molecules were captured by barcoded oligo-dT primers attached to the gel beads. No bulk RNA extraction was performed prior to library construction. Title: 10x Genomics Chromium Single Cell 3′ v3 library construction with CellPlex multiplexing Description: Libraries were generated using the 10x Genomics Chromium Single Cell 3′ Reagent Kit v3 in combination with the 3′ CellPlex Kit, according to the manufacturer's instructions. Eight biological samples were labelled with cell multiplexing oligos (CMO301, CMO302, CMO304–CMO309; CMO303 was not used) and pooled prior to GEM generation. Reverse transcription within GEMs tagged each cDNA and CMO molecule with a unique cell barcode and UMI. Following cDNA amplification, the amplified material was split to construct two separate libraries: a 3′ gene-expression (GEX) library, built by fragmentation, end repair, A-tailing, adapter ligation and sample-index PCR; and a multiplexing-capture (CP) library, generated from the CMO-derived fraction by sample-index PCR. Both libraries were quantified by Bioanalyzer and qPCR prior to sequencing.