Method Article

Single-cell RNA Sequencing for Profiling Ganglionic and Aganglionic Colonic Segments from Patients with Hirschsprung Disease

DOI:

10.3791/69825

⸱

May 22nd, 2026

 ,  ,  ,  ,  ,  ,  ,  ,  , 
1Department of Gastrointestinal Surgery, The Second Affiliated Hospital of Zunyi Medical University

In This Article

Summary

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This protocol describes a single-cell RNA sequencing workflow to analyze immune-cell infiltration and cell-type-specific transcriptional differences between ganglionic and aganglionic colonic segments in Hirschsprung disease.

Abstract

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Hirschsprung disease (HSCR) is a congenital intestinal motility disorder characterized by the absence of enteric neurons in the distal bowel. Compared with bulk RNA sequencing, single-cell RNA sequencing (scRNA-seq) enables gene expression profiling at the single-cell level, resolving cellular heterogeneity that is masked in population-averaged analyses. A standardized scRNA-seq workflow was established to characterize the transcriptional landscape of ganglionic and aganglionic segments from patients with HSCR. Colon tissues were collected, and hematoxylin and eosin staining was performed to identify ganglionic and aganglionic regions. Tissues were enzymatically and mechanically dissociated into single-cell suspensions, followed by filtration, viability assessment, and construction of single-cell transcriptome libraries. Libraries that passed quality control were sequenced, and raw data were processed for cell quantification and quality filtering to remove low-quality cells and technical artifacts. After normalization and dimensionality reduction, cells were clustered and annotated based on established marker genes. Downstream analyses compared cell-type composition between ganglionic and aganglionic tissues and identified differentially expressed genes within key immune cell subsets. The results reveal substantial differences in the immune microenvironment between ganglionic and aganglionic segments in HSCR.

Introduction

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Hirschsprung disease (HSCR) is one of the most common congenital intestinal motility disorders, with an incidence of approximately 1 in 5,000 live births worldwide1. The disease arises from the failure of enteric neural crest cells (ENCCs) to migrate into the distal intestine during embryonic weeks 5–12, resulting in the absence of enteric ganglia in the affected segments2. The aganglionic segment consequently exhibits persistent spastic contraction and impaired peristalsis, leading to proximal intestinal dilation. Clinically, this manifests as functional obstruction, causing constipation, abdominal distension, and vomiting, and may be life-threatening in severe cases. Surgical resection of the aganglionic segment remains the primary treatment for HSCR; however, a substantial proportion of patients experience postoperative complications, including constipation, enterocolitis, and fecal soiling, which significantly compromise quality of life3. Additionally, short bowel syndrome (SBS) may occur following extensive intestinal resection, resulting in impaired nutrient absorption and increased clinical management challenges. Therefore, elucidation of the underlying mechanisms of HSCR and identification of novel therapeutic targets remain critical clinical needs.

In recent years, single-cell RNA sequencing (scRNA-seq) has been widely applied to investigate disease-associated molecular mechanisms. Unlike conventional bulk RNA sequencing, scRNA-seq enables gene expression analysis at single-cell resolution, allowing the identification of cellular heterogeneity and rare subpopulations that are masked in bulk analyses4. Consequently, scRNA-seq has emerged as a powerful tool for dissecting cellular heterogeneity and intercellular communication, advancing research in biology and medicine5. This approach has revealed novel pathogenic mechanisms and therapeutic targets in complex diseases, including Alzheimer’s disease6, rheumatoid arthritis7, lung cancer8, and type 2 diabetes9.

In this study, scRNA-seq was applied to characterize the immune-cell transcriptional landscape and infiltration patterns in ganglionic and aganglionic colonic tissues from patients with HSCR. The workflow comprised three major steps: preparation and quality assessment of single-cell suspensions, construction of 3′ single-cell RNA-seq libraries, and downstream bioinformatic analysis. Tissue samples were preserved in a dedicated single-cell preservation solution immediately after resection and were enzymatically dissociated into single-cell suspensions within 48 h. Cell viability exceeded 80%, and the final cell concentration was adjusted to 700–1,500 cells/µL prior to library preparation. Immune cell infiltration between ganglionic and aganglionic segments was compared, and immune cell subpopulations exhibiting pronounced transcriptional differences were identified. These analyses delineate immune-cell alterations associated with colonic pathology in HSCR.

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Protocol

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This study was approved by the Institutional Ethics Committee of the Affiliated Hospital of Zunyi Medical University (Approval No.: KYLL-2024-030). The study complies with the Declaration of Helsinki. Informed consent (oral and written) was obtained from all participants and their guardians. Table 1 presents the information on HSCR patients.

Collect colonic tissues, including ganglionic and aganglionic segments, from HSCR patients diagnosed and undergoing surgery in the Department of Pediatric Surgery at the Affiliated Hospital of Zunyi Medical University. Include ganglionic and aganglionic segments from two male HSCR patients. Detailed clinical information is provided in Table 1. All the reagents, chemicals, equipment, and software are listed in the Table of Materials.

1. Hematoxylin and eosin staining

  1. Tissue collection and fixation
    1. Fix intestinal tissue samples in 4% paraformaldehyde fixation solution for at least 24 h.
      CAUTION: Paraformaldehyde is toxic and releases formaldehyde vapors. Perform all procedures in a chemical fume hood and use appropriate personal protective equipment.
    2. Trim tissues to approximately 5 × 5 × 2 mm3 using a scalpel. Place tissues into embedding cassettes and rinse under running tap water for 24 h.
      NOTE: Remove tissue edges and prepare blocks with at least one flat surface. If uneven, place tissue between filter papers after rinsing.
  2. Graded ethanol dehydration
    1. Immerse tissues sequentially in ethanol solutions: 70% ethanol overnight, 80% ethanol for 3 h, 90% ethanol for 2 h, 95% ethanol I for 2 h, 95% ethanol II for 1 h, 100% ethanol I for 1 h, and 100% ethanol II for 30 min.
      NOTE: Dilute ethanol (≥99.7%) with ultrapure water to the indicated concentrations.
      CAUTION: Perform ethanol handling in a chemical fume hood.
    2. Remove the dehydration basket and drain thoroughly between steps.
  3. Clearing
    1. Transfer tissues to xylene for 30 s to achieve transparency.
  4. Paraffin infiltration and embedding
    1. Melt paraffin wax at 60 °C one day in advance. Subject fresh wax to three freeze–thaw cycles to remove bubbles. Filter the melted paraffin through filter paper after the third cycle.
    2. Infiltrate tissues in pure paraffin wax (Paraffin wax I) for 1 h.
    3. Infiltrate tissues in paraffin mixture (Paraffin wax II; pure paraffin wax: beeswax = 8:1) for 2 h.
      NOTE: Completely melt beeswax and filter before use.
    4. Pour paraffin wax II into embedding molds. Place tissue blocks upright and flat, insert labels, and allow to solidify.
  5. Sectioning
    1. Trim paraffin blocks to obtain smooth surfaces without cutting into the tissue.
    2. Mount blocks onto a microtome. Adjust orientation and remove excess paraffin. Cut serial sections at 4 µm thickness. Float sections on a 50 °C water bath and transfer onto glass slides.
    3. Dry slides in a 60 °C oven to remove residual moisture.
      NOTE: Dry sections for 12–48 h.
  6. Deparaffinization
    1. Perform sequential incubation: xylene I (15 min), xylene II (15 min), 100% ethanol I (5 min), 100% ethanol II (5 min), 90% ethanol (5 min), 80% ethanol (5 min), 70% ethanol (5 min), and distilled water (5 min).
  7. Hematoxylin staining
    1. Stain sections with hematoxylin for 50 s.
    2. Rinse under running tap water for 10 min.
  8. Eosin staining
    1. Stain sections with eosin for 40 s.
  9. Dehydration
    1. Immerse sections sequentially in 95% ethanol I (30 s), 95% ethanol II (30 s), 100% ethanol I (30 s), and 100% ethanol II (30 s).
  10. Clearing
    1. Transfer sections into xylene I for 10 min and xylene II for 10 min.
  11. Mounting and observation
    1. Add mounting medium. Cover with a glass coverslip and remove air bubbles.
    2. Allow the mounting medium to solidify. Examine sections under a light microscope.

2. Single-cell RNA sequencing

  1. Preparation of single-cell suspension
    1. Mince intestinal tissues into ~1 mm3 fragments using scissors.
    2. Incubate tissue fragments in Dulbecco’s Modified Eagle Medium (DMEM) containing 1 mg/mL collagenase II and 0.1 mg/mL DNase I at 37 °C for 30 min with gentle agitation on a rocking platform (60 rpm).
    3. Terminate digestion by adding an equal volume of cold DMEM containing 10% fetal bovine serum (FBS).
    4. Filter the suspension through a 70 µm cell strainer. Centrifuge at 300 × g for 5 min at 4 °C.
    5. Discard the supernatant and retain the cell pellet. Wash once with phosphate-buffered saline (PBS) containing 0.04% bovine serum albumin (BSA).
    6. Dissociate cell clumps by gentle pipetting to obtain a single-cell suspension.
    7. Filter the suspension through a 40 µm cell strainer.
    8. Centrifuge at 300 × g for 5 min at 4 °C. Discard the supernatant and collect the pellet.
    9. Resuspend cells in DMEM containing 10% FBS and 1% penicillin–streptomycin (P/S).
    10. Determine cell viability and density using trypan blue exclusion. Use samples with viability >80% and adjust concentration to 700–1,500 cells/µL.
      NOTE: Mix cell suspension with trypan blue (1:1) and count viable cells using a hemocytometer.
  2. Library preparation
    1. Load approximately 15,000 cells per run for library preparation.
      NOTE: In gel bead-in-emulsion (GEM) microdroplets, mRNA hybridizes to poly(dT) oligonucleotides on Gel Beads.
    2. Perform reverse transcription and amplify cDNA using PCR (95 °C for 10 min; 40 cycles of 95 °C for 15 s, 60 °C for 35 s, 72 °C for 25 s; final extension at 72 °C for 25 s). Use amplified cDNA for 3′ scRNA-seq library construction.
      NOTE: A CCC overhang is added during cDNA synthesis and pairs with the template switching oligonucleotide (TSO) sequence.
    3. Break emulsions and recover cDNA from GEMs.
  3. Library QC and sequencing
    1. Quantify gene expression and assess fragment size distribution.
    2. Perform accurate quantification using a fluorescent qPCR instrument.
      NOTE: Ensure library concentration ≥10 nM.
    3. Sequence libraries using a sequencing instrument.
      NOTE: Read structure: Read 1 (26 bp), Read 2 (91 bp), i7 index (8 bp); target depth ~50,000 reads per cell.
  4. Data QC
    1. Align raw reads to the reference database. Perform UMI counting and generate a cell-by-gene matrix.
    2. Import results in AnnData (Annotated Data) format for downstream analysis.
    3. Perform QC filtering based on UMIs, gene counts, and mitochondrial reads.
      NOTE: Remove doublets (Scrublet); exclude cells with <200 or >5,000 genes, total_counts >40,000, or mt_ratio >20%.
  5. Dimensionality reduction and annotation
    1. Normalize Unique Molecular Identifier (UMI) counts to 10,000 per cell.
    2. Identify highly variable genes.
    3. Perform principal component analysis (PCA).
    4. Construct a k-nearest neighbor graph.
    5. Perform UMAP embedding.
    6. Cluster cells using the Leiden algorithm in Scanpy (v1.9.3).
      NOTE: Use sc.pp.neighbors and sc.tl.leiden (resolution = 0.5).
    7. Annotate cell types using a tool designed for the automated, high-accuracy annotation of cell types in single-cell RNA sequencing (scRNA-seq) data.
  6. Differential analysis
    1. Perform differential gene expression analysis in R (v4.2.2) using Seurat (v4.3.0).
      NOTE: Use FindMarkers function with the Wilcoxon rank-sum test.
    2. Perform GO enrichment analysis using clusterProfiler (v4.8.1) with enrichGO.
      NOTE: Includes CC, MF, and BP categories.
    3. Perform KEGG pathway enrichment analysis using clusterProfiler (v4.8.1) with enrichKEGG10.

3. Waste disposal

  1. Collect paraformaldehyde- and xylene-containing waste separately.
  2. Neutralize acidic solutions when appropriate or dispose of as hazardous waste.
  3. Follow institutional environmental health and safety (EHS) regulations.

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Results

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Hematoxylin and eosin (HE) staining (Figure 1) revealed distinct histological differences between ganglionic and aganglionic segments. Compared with ganglionic segments, aganglionic segments exhibited increased mucosal exfoliation, a marked reduction in goblet cells and submucosal neurons, and blurred boundaries between the mucosal and muscular layers. In a subset of sections, tissue preservation was suboptimal, with partial epithelial detachment and disruption of tissue architecture, limiti...

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Discussion

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Single-cell RNA sequencing was applied to profile transcriptional features of surgically resected colonic tissues (ganglionic and aganglionic segments) from patients with Hirschsprung diseases4,5. Particular attention was given to the tissue dissociation strategy, as dissociation parameters influence cell recovery, cell-type representation, and RNA integrity. Enzymatic digestion conditions were optimized to balance sufficient tissue dissociation with preservation...

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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This work was supported by Guizhou Science and Technology Project (Guizhou Science and Technology Cooperation - Foundation - ZK - [2024] General 344) (to X.F. Yang) and Zunyi Science and Technology Project Contract (Science and Technology Cooperation) (Zunyi Science and Technology Cooperation HZ (2023) No.) (to Y. Qu).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
BeeswaxSinopharm Chemical Reagent CO., Ltd.8012-89-3 https://www.reagent.com.cn/ProductSearch/8012-89-3
BeeswaxSinopharm Chemical Reagent CO., Ltd.8012-89-3 https://www.reagent.com.cn/ProductSearch/8012-89-3
Bovine serum albuminThermofisher30066575 https://www.thermofisher.cn/order/catalog/product/30066575?SID=srch-srp-30066575
Bovine serum albuminThermofisher30066575 https://www.thermofisher.cn/order/catalog/product/30066575?SID=srch-srp-30066575
CellTypistN/ANAhttp://celltypist.org/tutorials/onlineguide
CellTypistN/ANAhttp://celltypist.org/tutorials/onlineguide
Cluster profiler packageBioconductorv4.8.1https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html
Cluster profiler packageBioconductorv4.8.1https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html
Collagenase IIThermofisher17101015 https://www.thermofisher.cn/order/catalog/product/17101015?SID=srch-srp-17101015
Collagenase IIThermofisher17101015 https://www.thermofisher.cn/order/catalog/product/17101015?SID=srch-srp-17101015
Cover glassCITOTEST80340-0130 https://cn.citotest.com/pd40971476.html
Cover glassCITOTEST80340-0130 https://cn.citotest.com/pd40971476.html
Disposable microtome bladeFeatherS-35 https://www.feather.co.jp/en/m_Products/pathology01.html
Disposable microtome bladeFeatherS-35 https://www.feather.co.jp/en/m_Products/pathology01.html
DNase IThermofisher18047019 https://www.thermofisher.cn/order/catalog/product/18047019?SID=srch-srp-18047019
DNase IThermofisher18047019 https://www.thermofisher.cn/order/catalog/product/18047019?SID=srch-srp-18047019
Drying ovenBORUKDHG-9140A http://www.shboruike.com/sys-pd/84.html
Drying ovenBORUKDHG-9140A http://www.shboruike.com/sys-pd/84.html
Dulbecco’s Modified Eagle MediumThermofisher12491015 https://www.thermofisher.cn/order/catalog/product/12491015
Dulbecco’s Modified Eagle MediumThermofisher12491015 https://www.thermofisher.cn/order/catalog/product/12491015
Ethyl alcoholSinopharm Chemical Reagent CO., Ltd.10009218 https://www.reagent.com.cn/ProductSearch/10009218
Ethyl alcoholSinopharm Chemical Reagent CO., Ltd.10009218 https://www.reagent.com.cn/ProductSearch/10009218
Fetal bovine serumThermofisherA5669801https://www.thermofisher.cn/order/catalog/product/A5669801
Fetal bovine serumThermofisherA5669801https://www.thermofisher.cn/order/catalog/product/A5669801
Hematoxylin and eosin staining kitBeyotimeC0105S https://m.beyotime.com/mobilegoods.do?method=code&code=C0105S
Hematoxylin and eosin staining kitBeyotimeC0105S https://m.beyotime.com/mobilegoods.do?method=code&code=C0105S
Hydrochloric acidSinopharm Chemical Reagent CO., Ltd.10011028 https://www.reagent.com.cn/ProductSearch/10011028
Hydrochloric acidSinopharm Chemical Reagent CO., Ltd.10011028 https://www.reagent.com.cn/ProductSearch/10011028
Microscope slideCITOTEST80302-3111 https://cn.citotest.com/pd530614548.html
Microscope slideCITOTEST80302-3111 https://cn.citotest.com/pd530614548.html
Mounting mediumBeyotimeC0173-100ml https://m.beyotime.com/mobilegoods.do?method=code&code=C0173-100ml
Mounting mediumBeyotimeC0173-100ml https://m.beyotime.com/mobilegoods.do?method=code&code=C0173-100ml
Optical microscopeNikonECLIPSE SI https://www.microscope.healthcare.nikon.com/ja_JP/products/upright-microscopes/eclipse-si
Optical microscopeNikonECLIPSE SI https://www.microscope.healthcare.nikon.com/ja_JP/products/upright-microscopes/eclipse-si
Paraffin wax Sinopharm Chemical Reagent CO., Ltd.8002-74-2 https://www.reagent.com.cn/ProductSearch/8002-74-2
Paraffin wax Sinopharm Chemical Reagent CO., Ltd.8002-74-2 https://www.reagent.com.cn/ProductSearch/8002-74-2
Paraformaldehyde Fix SolutionBeyotimeP0099-3L https://m.beyotime.com/mobilegoods.do?method=code&code=P0099-3L
Paraformaldehyde Fix SolutionBeyotimeP0099-3L https://m.beyotime.com/mobilegoods.do?method=code&code=P0099-3L
Penicillin–streptomycinThermofisher15140122 https://www.thermofisher.cn/order/catalog/product/15140122?SID=srch-srp-15140122
Penicillin–streptomycinThermofisher15140122 https://www.thermofisher.cn/order/catalog/product/15140122?SID=srch-srp-15140122
Phosphate-buffered salineThermofisher10010023 https://www.thermofisher.cn/order/catalog/product/10010023
Phosphate-buffered salineThermofisher10010023 https://www.thermofisher.cn/order/catalog/product/10010023
Qualitative filter paperSolarbioYA0166https://www.solarbio.com
Qualitative filter paperSolarbioYA0166https://www.solarbio.com
R software packageR Foundation for Statistical Computingv4.2.2https://www.r-project.org/
R software packageR Foundation for Statistical Computingv4.2.2https://www.r-project.org/
Rotary MicrotomeLeicaLeica RM2125 RTShttps://www.leicabiosystems.com/en-jp/histology-equipment/microtomes/histocore-biocut/
Rotary MicrotomeLeicaLeica RM2125 RTShttps://www.leicabiosystems.com/en-jp/histology-equipment/microtomes/histocore-biocut/
ScanpyCommunity-developedv1.9.3https://scanpy.readthedocs.io/en/latest/?utm_source=chatgpt.com
ScanpyCommunity-developedv1.9.3https://scanpy.readthedocs.io/en/latest/?utm_source=chatgpt.com
SeuratCRANv4.3.0https://cran.r-project.org/web/packages/Seurat/index.html
SeuratCRANv4.3.0https://cran.r-project.org/web/packages/Seurat/index.html
Tissue dehydratorWuhan Junjie Electronics Co., LTDJT-12H http://www.whjjbl.com/front/page/126495/product_id/46166.html
Tissue dehydratorWuhan Junjie Electronics Co., LTDJT-12H http://www.whjjbl.com/front/page/126495/product_id/46166.html
Tissue embedding machineWuhan Junjie Electronics Co., LTDJB-L8 http://www.whjjbl.com/front/page/126495/product_id/46064.html
Tissue embedding machineWuhan Junjie Electronics Co., LTDJB-L8 http://www.whjjbl.com/front/page/126495/product_id/46064.html
Trypan blue SolarbioC0040https://www.solarbio.com/haveRegion?id=48&all=0&key=%20Trypan%20Blue
Trypan blue SolarbioC0040https://www.solarbio.com/haveRegion?id=48&all=0&key=%20Trypan%20Blue
XyleneSinopharm Chemical Reagent CO., Ltd.10023418 https://www.reagent.com.cn/ProductSearch/10023418
XyleneSinopharm Chemical Reagent CO., Ltd.10023418 https://www.reagent.com.cn/ProductSearch/10023418

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Tags

Single Cell RNA SequencingHirschsprung DiseaseGanglionic ColonAganglionic ColonGene Expression ProfilingEnteric NeuronsCell Type CompositionImmune MicroenvironmentTissue DissociationDifferential Gene Expression

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