Murine intestinal cell dissociation suitable for multi-omics single-cell assays v1

Here we present an optimized protocol for the single-cell suspension of murine intestines (here we used ileum) optimized for contemporary multi-omics single-cell assays including high-dimensional flow cytometry and single-cell RNA-seq. This protocol provides a rapid and efficient method of digesting mouse ileum into a single-cell suspension with a short processing time, high viable cell yield, and it preserves cell-surface markers throughout the process. Notably, the protocol utilizes a Collagenase-I/Benzonase-based 37°C water bath dissociation method and collects already digested cells at several steps during the process to prevent over-digestion (fractioning technique). Tissues were normalized by length (2.5 cm) and weight (averaged 79.5 mg) and measured for cell yield and viability. We obtained an average total cell yield of ~4.4E6 +/- 1.4E6 cells, and average viability of ~56 +/- 13% (n=7) (Figure 1). By flow cytometry, ~30%-50% of cells were CD45+ immune cells, with the remainder of the cell population being CD45- non-immune cells (i.e., epithelial and stromal cells). To optimize this protocol, several other conditions were tested, including different digestive enzymes, mechanical digestion techniques, the inclusion of an intraepithelial layer (IEL) digestion, and an antioxidant cocktail addition. We summarize our main findings about the optimization of this protocol below: Collagenase-I provided the highest number of total yield and viable cells when compared to Collagenase-IV and Liberase. Enzymatic digestion in a 37°C waterbath and vortexing the cell solutions proved more successful in cell yield and viability than different combinations of the gentleMACS Tissue Dissociator (Myltenyi) intestine digestion programs, including its m_ intestine _1 and 37_m_LPDK programs and variations in their run times. We have also validated a protocol to separate the IEL before the rest of the ileum, and have found it compatible and therefore able to be adopted with the recommended protocol while maintaining a similar yield of viable cells. An IEL separation protocol has been included in a separate section at the end. By using our "fractioning technique", in which cells that are digested early on are removed before digesting the rest of the tissue separately, total cell yield and viability are increased. We tested several antioxidant combinations (Gluthatione, Vitamin C, and Vitamin E) and found higher live cell yield when excluding these antioxidants from the Collagenase-I digestion step. Based on these findings, this murine intestinal digestion protocol is rapid and efficient and can be used for multi-omics single-cell assays such as high-dimensional flow cytometry and single-cell RNA-sequencing.