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

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.