Processing Human Colon muscle layers for Single Nuclei RNA-seq using PHOX2B antibody immunoselection v1
Year: 2019
Type: preprint
Abstract: ABSTRACT INTRODUCTION: The human enteric nervous system (ENS) is a complex network of neurons and glia that extends throughout the length of the bowel. There are many neuron and glia types, but very little is known about the human ENS compared to other species. There are two main ENS layers. The myenteric plexus neurons are clustered into ganglia with thick nerve fiber bundles running between ganglia. Submucosal neuron ganglia are scattered throughout the region between circular muscle and the epithelial lining of the bowel. One key problem with studying human ENS is that these cells are a very minor component of the bowel wall (< 1: 10,000 cells) and they are surrounded by other tissue. The bowel wall is not easily dissociated making it challenging to isolate cells of the human ENS for any type of analsysis. OBJECTIVES: To enrich for neurons from human colon myenteric plexus using PHOX2B antibody and FACS METHODS: 1) Human colon muscle was dissected from the bowel wall and then frozen in OCT. 2) Frozen muscle was sectioned on a cryostat to generate small fragments and disrupt muscle and connective tissue. 3) RNA quality was assessed from a few sections using an Agilent Bioanalyzer. 4) Nuclei were obtained from frozen sections using Dounce homogenization and stained with antibody to PHOX2B. 5). Nuclei were separated from other cell debris by FACS. 6) RNA-seq data were obtained using the 10X Genomic single cell sequencing platform.RESULTS: This procedure worked well to isolate nuclei from human colon muscle. PHOX2B antibody labeling led to a small population of nuclei that appeared to be antibody stained under the microscope and have attached antibody by FACS. Unfortunately, single nucleus RNA-seq demonstrated that we had not enriched for myenteric neurons with this approach. RNA-seq data were obtained from many other cell types within the muscle layers of the bowel wall.CONCLUSION: This approach generated valuable data at a single nucleus RNA-seq level for many cell types within the muscle wall known to control bowel motility, but did not enrich for neuronal nuclei, even though PHOX2B antibody selectively stains myenteric plexus nuclei within the human bowel myenteric plexus
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3137513995