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URL: https://dx.doi.org/DOI:10.17504/protocols.io.bhbkj2kw

Authors: Benjamin Izar, Parin Shah, Mei-Ju Su, Isaac Wakiro, Sara Napolitano, Jingyi Wu, Sébastien Vigneau, Asaf Rotem, Orit Rozenblatt-Rosen, Bruce Johnson, Aviv Regev

Group: NCIHTAN

Summary: This protocol is used to process human ovarian cancer ascites to a single-cell suspension compatible with droplet-based single-cell RNA-Seq technology.A majority of ascites samples have a high proportion (>90%) of CD45-expressing (CD45+) cells, thus greatly reducing the ability to capture non-immune and malignant cells. To address this issue, we have included a CD45+ cell depletion step to reduce the number of CD45+ cells. In such samples, where the proportion of immune cells is very high, a large proportion of both CD45+ and CD45- cells can be recovered post-depletion, allowing simultaneous assessment of the immune and non-immune microenvironment. In cases with a lower initial proportion of CD45+ cells, however, non-immune cells tend to be dominant following CD45+ cell depletion.Of note, many ascites samples contain multicellular aggregates, or spheroids, which pose challenges during sample processing and dissociation. Larger aggregates are removed through an initial 70 μm straining (Step 14) whereas smaller residual aggregates, when present, can be removed through a final 30 μm straining (optional Step 20). Yet, we have encountered instances where small aggregates cannot be efficiently removed through straining. This protocol is not optimized for those cases. In addition, we would like to note that ascites can be quite variable and further optimization of this protocol may be needed to handle cases with atypical characteristics. For the Human Tumor Atlas Pilot Project (HTAPP), this protocol was successfully applied to 4 ovarian cancer ascites samples. Description of this protocol and guidance for testing and selecting methods for processing other tumor and sample types can be found in Slyper et al.

Affiliations: Dana-Farber Cancer Institute;Human Tumor Atlas Pilot Project, Dana-Farber Cancer Institute;Human Tumor Atlas Pilot Project, Dana-Farber Cancer Institute;Human Tumor Atlas Pilot Project, Dana-Farber Cancer Institute, University of California, Santa Barbara, Dana-Farber Cancer Institute;Human Tumor Atlas Pilot Project, Dana-Farber Cancer Institute;Human Tumor Atlas Pilot Project, Dana-Farber Cancer Institute;Human Tumor Atlas Pilot Project, Broad Institute;Human Tumor Atlas Pilot Project, Dana-Farber Cancer Institute;Human Tumor Atlas Pilot Project, Broad Institute;Human Tumor Atlas Project;Massachusetts Institute of Technology;Howard Hughes Medical Institute

Version: 2

Publication Date: 2020

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