Single cell dissociation of intestinal gut tissue for scRNAseq_v2 v1

This protocol was developed for scRNAseq of intestinal biopsies. For our study, we needed to preserve all cell types and minimise changes to gene expression, while keeping all cells healthy in order to detect small genotype-associated changes in gene expression (expression quantitative trait loci). To achieve this, we use the Bacillus Licheniformis Protease (BLP) which digests intestinal biopsies on ice. BLP does not release mesenchymal cells very well, and so Collagenase IV is used at RT for 10 min to release more of those. We add two inhibitors of anoikis - Rocki and a pan caspase inhibitor - at the start of the digest. No digests are performed at 37oC to avoid anoikis and minimise gene expression changes. Finally, cells are incubated in a hypotonic solution to remove low quality cells that are pro-apoptotic. Expected results: Trypan blue staining shows cell viability is comparable to Collagenase digests (90%). It takes another ~1 h for cells to pass through the Chromium. After 10X, we see a substantial increase in the viability of epithelial cells compared to warm collagenase digest methods. The digest supports the isolation of healthy intestinal epithelial cells, which are usually of low quality as they are prone to anoikis during standard EDTA incubation or warm collagenase digests. All major cell types are identified, including rare tuft cells and enteroendocrine cells. Although detectable, mesenchymal cells are not as abundant as they are after warm collagenase digests.