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INTESTINAL Publication-derived

Intestinal Organoids (2016, No. 2)

Full SOP with Academic or a plan

Source Blokzijl et al. · Hubrecht Institute and University Medical Centre Utrecht, Utrecht, Netherlands · 10.1038/nature19768

Authors: Blokzijl F, de Ligt J, Jager M, Sasselli V, Roerink S, Sasaki N, Huch M, Boymans S, Kuber E, Torrber P, de Bruijn E, Clevers H, Cuppen E, Stratton MR Duration: 450 days 9 phases Cell source: Primary (Human Intestinal Biopsies, Age Range 3-87 Years)

Abstract

Used clonal organoid expansion and whole-genome sequencing to quantify somatic mutation accumulation in human intestinal stem cells across the human lifespan. Found approximately 40 mutations per stem cell per year with clock-like signatures (SBS1, SBS5), establishing the baseline somatic mutational landscape of normal intestine.

Cell source
Primary (Human Intestinal Biopsies, Age Range 3-87 Years)
Application
Aging Biology

Protocol overview

28 steps across 9 phases

Phase 1 · Human tissue acquisition and processing Day 0-1
  1. a Obtain endoscopic biopsies
  2. b Obtain liver transplant biopsies
  3. c Obtain tumour-adjacent colon tissue
Phase 2 · Crypt dissociation and bulk culture Day 1-14
  1. a Dissociate intestinal crypts from biopsy samples
  2. b Isolate and culture hepatic cells from liver biopsies
Phase 3 · Single-cell isolation and clonal expansion Day 8-56
  1. a Sort single cells by flow cytometry
  2. b Expand single cells into clonal organoid cultures
  3. c Collect clonal organoid cultures
Phase 4 · Genomic DNA extraction Day 50-57
  1. a Extract genomic DNA from clonal organoid cultures
  2. b Prepare reference samples (blood or biopsy)
Phase 5 · Library preparation and whole-genome sequencing Day 57-70
  1. a Prepare Illumina sequencing libraries
  2. b Perform paired-end whole-genome sequencing
  3. c Align reads to reference genome
  4. d Mark duplicates and perform realignment
Phase 6 · Variant calling and filtering Day 70-85
  1. a Call variants using GATK UnifiedGenotyper
  2. b Apply initial variant quality filters
  3. c Filter variants to obtain high-quality somatic mutations
  4. d Validate clonal origin using variant allele frequency analysis
Phase 7 · Independent validation of mutation calls Day 80-100
  1. a Design amplicon primers for validation
  2. b Perform PCR amplification of candidate regions
  3. c Prepare amplicon sequencing libraries
  4. d Sequence amplicons on MiSeq platform
  5. e Validate variant calls
Phase 8 · Assessment of in vitro-induced mutations (optional extended culture) Day 300-450 (optional)
  1. a Perform extended in vitro culture
  2. b Perform second clonal expansion from extended cultures
Phase 9 · Mutational signature and structural variant analysis Day 100-150
  1. a Extract mutational signatures using NMF
  2. b Detect copy-number variations (CNVs)
  3. c Perform selection analysis (dN/dS ratio)

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Attribution

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

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