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PANCREAS Publication-derived

Enhancer Reprogramming Promotes Pancreatic Cancer Metastasis - Organoid Culture and Epigenomic Analysis Protocol

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

👤 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 ⏱ 360 days 📋 12 phases 🧫 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

Tissue Dissection and Organoid Establishment Days 1-14
  1. 1 Tissue Collection and Mincing
  2. 2 Enzymatic Digestion
  3. 3 TrypLE Secondary Digestion
  4. 4 Cell Resuspension and Matrigel Embedding
  5. 5 Pancreatic Organoid Media Preparation and Culture Initiation
  6. 6 Organoid Maturation and Passage
Organoid Dissociation and Cell Line Generation Days 14-28
  1. 7 Organoid Dissociation into Single Cells
  2. 8 KPC-2D Cell Line Generation from Tumor Organoids
  3. 9 Mycoplasma Testing and Genotyping
Chromatin Immunoprecipitation (ChIP) Days 28-35
  1. 10 Cell Harvesting and Crosslinking
  2. 11 Cell Lysis and Chromatin Isolation
  3. 12 Chromatin Sonication
  4. 13 Chromatin Clarification and Pre-clearing
  5. 14 Immunoprecipitation
  6. 15 Immunocomplex Washing
  7. 16 Chromatin Elution and Reverse-crosslinking
  8. 17 DNA Purification
ChIP-seq Library Construction and Sequencing Days 35-42
  1. 18 End-Repair and dA-Tailing
  2. 19 Adapter Ligation
  3. 20 Size Selection and Gel Extraction
  4. 21 PCR Amplification
  5. 22 Final Library Cleanup and QC
  6. 23 Library Quantification and Pooling for Sequencing
  7. 24 Illumina Sequencing
RNA-seq Library Construction Days 42-49
  1. 25 RNA Extraction
  2. 26 RNA Precipitation and Purification
  3. 27 RNA Quality Assessment
  4. 28 Poly-A Selection and Fragmentation
  5. 29 cDNA Synthesis and Library Construction
  6. 30 Library Quantification and QC
  7. 31 Illumina Sequencing
ATAC-seq Library Construction Days 49-56
  1. 32 Cell Harvesting and Lysis
  2. 33 Nuclei Isolation
  3. 34 Tn5 Transposition
  4. 35 Transposition Termination and DNA Recovery
  5. 36 PCR Amplification
  6. 37 Library Cleanup and QC
  7. 38 Library Pooling and Sequencing
Molecular Biology: FOXA1 Overexpression and Knockdown Days 56-84
  1. 39 cDNA Cloning and Construct Preparation
  2. 40 Retrovirus Production in Phoenix Cells
  3. 41 Retrovirus Concentration
  4. 42 Retroviral Infection of Organoids
  5. 43 Lentiviral shRNA Production and Infection
  6. 44 CRISPR-Cas9 Genome Editing in Organoids
  7. 45 Western Blotting Validation
In Vitro Phenotypic Assays Days 84-112
  1. 46 Anchorage-Independent Sphere Culture
  2. 47 Boyden Chamber Invasion Assay
  3. 48 Clonogenic Growth Assay
  4. 49 Cell Migration Assay (ORIS)
In Vivo Transplantation Assays Days 112-210
  1. 50 Orthotopic Pancreatic Injection
  2. 51 Tail Vein Injection
  3. 52 Portal Vein Injection
  4. 53 Subcutaneous Injection
  5. 54 Intraperitoneal Injection
  6. 55 Intrasplenic Injection
  7. 56 Tumor Monitoring and Endpoint Determination
Tissue Histology and Immunohistochemistry Days 210-240
  1. 57 Tissue Fixation and Processing
  2. 58 Hematoxylin & Eosin (H&E) Staining
  3. 59 Immunohistochemistry (IHC) for FOXA1
  4. 60 RNA Fluorescence In Situ Hybridization (RNA FISH)
  5. 61 Metastasis Quantification
  6. 62 Tumor Weight Quantification
Computational Analysis - RNA-seq Days 240-300
  1. 63 RNA-seq Alignment and Read Counting
  2. 64 Gene Expression Filtering and Normalization
  3. 65 Differential Gene Expression Analysis
  4. 66 Hierarchical Clustering and Heatmap Generation
  5. 67 Gene Set Enrichment Analysis (GSEA)
Computational Analysis - ChIP-seq and ATAC-seq Days 300-360
  1. 68 ChIP-seq and ATAC-seq Read Alignment
  2. 69 Peak Identification in ChIP-seq
  3. 70 GAIN Region Identification
  4. 71 Transcription Factor Motif Enrichment Analysis
  5. 72 Metagene and Heatmap Visualization
  6. 73 Quantitative Meta-profile Comparison
  7. 74 Gene Ontology Analysis

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Roe et al., 2017. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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