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PANCREAS Publication-derived

Pancreatic Ductal Adenocarcinoma Organoids

Full SOP with Academic or a plan

Source Roe et al., 2017 · Cold Spring Harbor Laboratory · 10.1016/j.cell.2017.07.007

Authors: Jae-Seok Roe, Chang-Il Hwang, Tim D.D. Somerville, Joseph P. Milazzo, Eun Jung Lee, Brandon Da Silva, Laura Maiorino, Hervé Tiriac, C. Megan Young, Koji Miyabayashi, Dea Filippini, Brianna Creighton, Richard A. Burkhart, Jonathan M. Buscaglia, Edward J. Kim, Jean L. Grem, Audrey J. Lazenby, James A. Grunkemeyer, Michael A. Hollingsworth, Paul M. Grandgenett, Mikala Egeblad, Youngkyu Park, David A. Tuveson, Christopher R. Vakoc Duration: 360 days 12 phases Cell source: Patient-Derived iPSC (Pancreatic Ductal Adenocarcinoma)

Abstract

This protocol establishes paired primary tumor and metastasis-derived organoid cultures from the KPC mouse model and human PDA specimens to characterize enhancer landscape evolution during pancreatic cancer progression. The workflow includes tissue dissociation, organoid culture, epigenomic profiling (ChIP-seq, ATAC-seq, RNA-seq), and in vivo transplantation assays to identify FOXA1-mediated enhancer reprogramming as a driver of metastatic traits.

Cell source
Patient-Derived iPSC (Pancreatic Ductal Adenocarcinoma)
Application
Disease modeling

Protocol overview

74 steps across 12 phases

Phase 1 · Tissue Dissection and Organoid Establishment Days 1-14
  1. a Tissue Collection and Mincing
  2. b Enzymatic Digestion
  3. c TrypLE Secondary Digestion
  4. d Cell Resuspension and Matrigel Embedding
  5. e Pancreatic Organoid Media Preparation and Culture Initiation
  6. f Organoid Maturation and Passage
Phase 2 · Organoid Dissociation and Cell Line Generation Days 14-28
  1. a Organoid Dissociation into Single Cells
  2. b KPC-2D Cell Line Generation from Tumor Organoids
  3. c Mycoplasma Testing and Genotyping
Phase 3 · Chromatin Immunoprecipitation (ChIP) Days 28-35
  1. a Cell Harvesting and Crosslinking
  2. b Cell Lysis and Chromatin Isolation
  3. c Chromatin Sonication
  4. d Chromatin Clarification and Pre-clearing
  5. e Immunoprecipitation
  6. f Immunocomplex Washing
  7. g Chromatin Elution and Reverse-crosslinking
  8. h DNA Purification
Phase 4 · ChIP-seq Library Construction and Sequencing Days 35-42
  1. a End-Repair and dA-Tailing
  2. b Adapter Ligation
  3. c Size Selection and Gel Extraction
  4. d PCR Amplification
  5. e Final Library Cleanup and QC
  6. f Library Quantification and Pooling for Sequencing
  7. g Illumina Sequencing
Phase 5 · RNA-seq Library Construction Days 42-49
  1. a RNA Extraction
  2. b RNA Precipitation and Purification
  3. c RNA Quality Assessment
  4. d Poly-A Selection and Fragmentation
  5. e cDNA Synthesis and Library Construction
  6. f Library Quantification and QC
  7. g Illumina Sequencing
Phase 6 · ATAC-seq Library Construction Days 49-56
  1. a Cell Harvesting and Lysis
  2. b Nuclei Isolation
  3. c Tn5 Transposition
  4. d Transposition Termination and DNA Recovery
  5. e PCR Amplification
  6. f Library Cleanup and QC
  7. g Library Pooling and Sequencing
Phase 7 · Molecular Biology: FOXA1 Overexpression and Knockdown Days 56-84
  1. a cDNA Cloning and Construct Preparation
  2. b Retrovirus Production in Phoenix Cells
  3. c Retrovirus Concentration
  4. d Retroviral Infection of Organoids
  5. e Lentiviral shRNA Production and Infection
  6. f CRISPR-Cas9 Genome Editing in Organoids
  7. g Western Blotting Validation
Phase 8 · In Vitro Phenotypic Assays Days 84-112
  1. a Anchorage-Independent Sphere Culture
  2. b Boyden Chamber Invasion Assay
  3. c Clonogenic Growth Assay
  4. d Cell Migration Assay (ORIS)
Phase 9 · In Vivo Transplantation Assays Days 112-210
  1. a Orthotopic Pancreatic Injection
  2. b Tail Vein Injection
  3. c Portal Vein Injection
  4. d Subcutaneous Injection
  5. e Intraperitoneal Injection
  6. f Intrasplenic Injection
  7. g Tumor Monitoring and Endpoint Determination
Phase 10 · Tissue Histology and Immunohistochemistry Days 210-240
  1. a Tissue Fixation and Processing
  2. b Hematoxylin & Eosin (H&E) Staining
  3. c Immunohistochemistry (IHC) for FOXA1
  4. d RNA Fluorescence In Situ Hybridization (RNA FISH)
  5. e Metastasis Quantification
  6. f Tumor Weight Quantification
Phase 11 · Computational Analysis - RNA-seq Days 240-300
  1. a RNA-seq Alignment and Read Counting
  2. b Gene Expression Filtering and Normalization
  3. c Differential Gene Expression Analysis
  4. d Hierarchical Clustering and Heatmap Generation
  5. e Gene Set Enrichment Analysis (GSEA)
Phase 12 · Computational Analysis - ChIP-seq and ATAC-seq Days 300-360
  1. a ChIP-seq and ATAC-seq Read Alignment
  2. b Peak Identification in ChIP-seq
  3. c GAIN Region Identification
  4. d Transcription Factor Motif Enrichment Analysis
  5. e Metagene and Heatmap Visualization
  6. f Quantitative Meta-profile Comparison
  7. g Gene Ontology Analysis

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Attribution

This SOP was written by Organthis based on the published method in Roe et al., 2017. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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