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BRAIN Publication-derived

Autism Spectrum Disorder Cortical Organoids with Single-Cell Profiling

Full SOP with Academic or a plan

Source Jang et al., 2023 · DGIST (Daegu Gyeongbuk Institute of Science and Technology) · 10.1038/s41380-022-01822-1

Authors: Wooyoung Eric Jang, Ji Hwan Park, Gaeun Park, Geul Bang, Chan Hyun Na, Jin Young Kim, Kwang-Youl Kim, Kwang Pyo Kim, Chan Young Shin, Joon-Yong An, Yong-Seok Lee, Min-Sik Kim Duration: 75 days 5 phases Cell source: Patient-Derived iPSC (CNTNAP2-associated ASD); Mouse Neural Tissue (Cntnap2 KO)

Abstract

This protocol integrates quantitative mass spectrometry-based proteometabolomic analysis of medial prefrontal cortex tissue from Cntnap2 knockout mice with publicly available multi-omics data from autism spectrum disorder (ASD) patient brains and CNTNAP2-mutant forebrain organoids. The workflow identifies Cntnap2-dependent molecular networks dysregulated in ASD, revealing alterations in mitochondrial function, synaptic vesicle transport, and axonal integrity through integrated data analysis and functional network modeling.

Cell source
Patient-Derived iPSC (CNTNAP2-associated ASD); Mouse Neural Tissue (Cntnap2 KO)
Application
Disease modeling; Multi-omics biomarker discovery for autism spectrum disorder

Protocol overview

23 steps across 5 phases

Phase 1 · Mouse Model Preparation and Tissue Dissection Day 1-14
  1. a Animal breeding and genotyping
  2. b Three-chamber social preference behavioral test
  3. c Medial prefrontal cortex tissue dissection
Phase 2 · Protein Extraction and Quantitative Proteomic Analysis Day 15-30
  1. a Protein extraction from mPFC tissue
  2. b Enzymatic digestion with trypsin
  3. c TMT 10-plex labeling of peptides
  4. d Pool labeled peptides and reversed-phase liquid chromatography fractionation
  5. e High-resolution Orbitrap MS analysis in data-dependent acquisition mode
Phase 3 · Targeted Quantitative Metabolomic Analysis Day 31-45
  1. a Metabolite extraction from mPFC tissue
  2. b Internal standard spiking and aliquoting
  3. c LC–MS/MS analysis of amino acids and biogenic amines (Aliquot 1)
  4. d FIA-MS/MS analysis of lipid species (Aliquot 2)
Phase 4 · Differential Expression Analysis and Data Integration Day 46-60
  1. a Identification of differentially expressed peptides (DEPeptides) from proteomics data
  2. b Identification of differentially expressed metabolites (DEMs) from metabolomic data
  3. c Comparison with human ASD patient multi-omics datasets
  4. d Gene ontology (GO) and functional enrichment analysis
  5. e Single-cell RNA-seq reanalysis of CNTNAP2-mutant forebrain organoids
  6. f Cell-type-specific differential gene expression analysis
  7. g Protein-protein interaction network analysis
Phase 5 · Molecular Network Model Construction Day 61-75
  1. a Selection of pathway-relevant molecules
  2. b Network model 1: Myelin sheath and mitochondrial dysfunction
  3. c Network model 2: Synaptic vesicle dysfunction
  4. d Network model 3: Neuronal projection and growth cone dynamics

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Attribution

This SOP was written by Organthis based on the published method in Jang et al., 2023. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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