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URL: https://dx.doi.org/DOI:10.17504/protocols.io.bhbdj2i6

Authors: Eugene Drokhlyansky, Nicholas Van Wittenberghe, Michal Slyper, Julia Waldman, Asa Segerstolpe, Orit Rozenblatt-Rosen, Aviv Regev

Group: NCIHTAN

Summary: This protocol describes a nuclei isolation method for frozen tissues based on the previously published protocol by Gao et al. and modified by Drokhlyansky et al. It can be used on both healthy and disease tissues and is compatible with droplet-based single-nucleus RNA-Seq technology Slyper et al.This method is part of a toolbox for processing frozen tissue samples for single-nucleus RNA-Seq, including the NST (this protocol), CST and TST protocols (all available in protocols.io). We recommend users to test all three protocols on their tissue type of interest and perform side-by-side comparison of the data generated.  A description of the complete toolbox and guidance for testing and selecting methods from the toolbox for processing other tumors can be found in Slyper et al.For the Human Tumor Atlas Pilot Project (HTAPP), this protocol was tested on metastatic breast cancer, ovarian cancer and pediatric neuroblastoma frozen samples. For these cancer types, however, TST was the protocol of choice. When tested on HTAPP samples, NST, in general, underperformed compared to TST and CST based on several QC parameters.

Affiliations: Broad Institute;Human Tumor Atlas Pilot Project, Broad Institute;Human Tumor Atlas Pilot Project, Broad Institute;Human Tumor Atlas Pilot Project, Broad Institute;Human Tumor Atlas Pilot Project, Broad Institute;Human Tumor Atlas Pilot Project, Broad Institute;Human Tumor Atlas Pilot Project, Broad Institute;Human Tumor Atlas Pilot Project;Massachusetts Institute of Technology;Howard Hughes Medical Institute

Version: 2

Publication Date: 2020

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