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INTESTINAL Publication-derived

Colonic Organoids for Disease Modelling

Full SOP with Academic or a plan

Source Drost et al. · Hubrecht Institute and University Medical Centre Utrecht, Utrecht, Netherlands · 10.1126/science.aao3130

Authors: Drost J, van Boxtel R, Blokzijl F, Mizutani T, Sasaki N, Duursma A, de Ligt J, Offerhaus GJ, Begthel H, Korber J, van de Wetering M, Schwank G, Clevers H Duration: 120 days 6 phases Cell source: Patient-Derived (FAP Patient Normal and Adenoma Tissue)

Abstract

Used organoids from familial adenomatous polyposis patients to demonstrate that APC-heterozygous normal intestinal epithelium accumulates mutations at an accelerated rate compared to wild-type tissue. Whole-genome sequencing revealed distinct mutational signatures active during early tumorigenesis.

Cell source
Patient-Derived (FAP Patient Normal and Adenoma Tissue)
Application
Cancer Prevention

Protocol overview

25 steps across 6 phases

Phase 1 · CRISPR-Cas9 Gene Targeting and Knockout Generation Days 1-14
  1. a Design and Prepare CRISPR Components
  2. b Transfect Organoids with CRISPR-Cas9 and Selection Cassette
  3. c Puromycin Selection
  4. d Clonal Expansion and Single Organoid Isolation
  5. e Genotyping by PCR and Sequencing
  6. f Verify Gene Inactivation by qRT-PCR
  7. g Confirm Protein Loss by Western Blot
Phase 2 · Mutation Accumulation Period and Subclonal Derivation Days 15-90 (MLH1) or Days 15-120 (NTHL1)
  1. a Passage Knockout and Parental Organoids
  2. b Generate Subclonal Cultures via Flow Cytometry
  3. c Expand Subclonal Organoids
Phase 3 · DNA Extraction and Whole-Genome Sequencing Days 91-120+
  1. a Isolate Genomic DNA
  2. b Perform Whole-Genome Sequencing (WGS)
  3. c Align Sequencing Reads and Call Variants
  4. d Filter and Annotate Mutations
  5. e Calculate Mutation Accumulation Rates
Phase 4 · Mutational Signature Extraction and Analysis Post-sequencing
  1. a Extract Mutational Spectra
  2. b Compare to COSMIC Signatures
  3. c Analyze Indel Patterns
Phase 5 · Replication Timing and Genomic Distribution Analysis Post-sequencing
  1. a Perform Replication Timing Analysis (Repli-seq)
  2. b Determine Replication Origin Locations
  3. c Correlate Mutations with Replication Timing
  4. d Analyze Replication Strand Asymmetry
Phase 6 · Validation and Comparison to Clinical Samples Post-analysis
  1. a Obtain Clinical Cancer Sequencing Data
  2. b Perform Cosine Similarity Analysis on Clinical Samples
  3. c Screen for Germline Mutations in Candidate Genes

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Attribution

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

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