| Autor: |
Astrid Kosters, Junkai Yang, Ann Dodd, Mackenzie L White, Greg Gibson, Subra Kugathasan, Peng Qiu, Eliver Ghosn |
| Rok vydání: |
2021 |
| Předmět: |
|
| DOI: |
10.17504/protocols.io.bubrnsm6 |
| Popis: |
We developed a protocol for the preparation of a single-cell suspension of human intestinal (ileal) biopsies, optimized for contemporary multi-omics single-cell assays including high-dimensional flow cytometry and single-cell RNA sequencing (scRNA-seq) assays. The protocol provides a rapid and efficient method of digesting human intestinal biopsies into a single-cell suspension resulting in a high viable cell yield and preservation of cell-surface markers. Notably, the protocol utilizes a Collagenase-I/Benzonase-based 37°C dissociation method, which collects already digested cells at several steps during the process to prevent over-digestion (fractioning technique). We obtained a total cell yield of ~5.8E6 +/- 1.9E6 cells, with a viability of 71% +/- 6% (n=9) (Figures 1A and 1B) from biopsy weights ranging 10-14 mg. By flow cytometry, all major immune cell lineages were detected, as well as a CD45-/EpCAM+ epithelial cell population (20%-60% of total viable singlets; Figure 1C). scRNA-seq analysis (10X Genomics; 5’/VDJ with feature barcoding) showed high-quality viable cells with low mitochondrial gene counts, indicating this protocol exerts minimal stress on the digested cells (Figure 1D). |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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