Human Tumor Atlas Pilot Project (HTAPP) Webinar Series

LINKS

Toolbox paper

Slyper et al Nature Medicine volume 26, pp 792–802(2020): A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors.

Abstract

Single-cell genomics is essential to chart tumor ecosystems. Although single-cell RNA-Seq (scRNA-Seq) profiles RNA from cells dissociated from fresh tumors, single-nucleus RNA-Seq (snRNA-Seq) is needed to profile frozen or hard-to-dissociate tumors. Each requires customization to different tissue and tumor types, posing a barrier to adoption. Here, we have developed a systematic toolbox for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq, respectively. We analyzed 216,490 cells and nuclei from 40 samples across 23 specimens spanning eight tumor types of varying tissue and sample characteristics. We evaluated protocols by cell and nucleus quality, recovery rate and cellular composition. scRNA-Seq and snRNA-Seq from matched samples recovered the same cell types, but at different proportions. Our work provides guidance for studies in a broad range of tumors, including criteria for testing and selecting methods from the toolbox for other tumors, thus paving the way for charting tumor atlases.

Protocols

Protocols.io links to protocols discussed in the webinar and other protocols included in Slyper et al are provided below.

Nuclei isolation from frozen tissue for single-nucleus RNA-Seq

1. HTAPP_NST- Nuclei isolation from frozen tissue V.2
2. HTAPP_TST- Nuclei isolation from frozen tissue V.2
3. HTAPP_CST- Nuclei isolation from frozen tissue V.2

Fresh tissue processing protocols for single-cell RNA-Seq

1. HTAPP_Dissociation of human metastatic breast cancer core needle biopsy to a single-cell suspension for single-cell RNA-seq V.2
2. HTAPP_Dissociation of human primary lung cancer resection to a single-cell suspension for single-cell RNA-seq V.2
3. HTAPP_Depletion of CD45+ cells from single-cell suspension for single-cell RNA-seq
4. HTAPP_Dissociation of human ovarian cancer resection to a single-cell suspension for single-cell RNA-seq V.2
5. HTAPP_Processing human ovarian cancer ascites to a single-cell suspension for single-cell RNA-seq V.2
6. HTAPP_Depletion of CD45+ cells from ovarian cancer ascites single cell suspensions for single-cell RNA-Seq V.2
7. HTAPP_Dissociation of human neuroblastoma tumors to a single-cell suspension for single-cell RNA-seq using Papain

WEBINAR 1: Tissue acquisition workflows and sc/snRNA-Seq processing protocols

December 3rd, 2020

AGENDA

1. HTAPP overview
2. Fresh tissue acquisition workflow
3. Key components of a tissue acquisition and allocation workflow
4. Key aspects to keep in mind for each component
5. Fresh tissue processing for sc-RNAseq
6. Description of the processing workflow
7. Guidelines for testing and selecting protocols
8. Frozen tissue processing for snRNA-Seq
9. Description of the processing workflow
10. Guidelines for testing and selecting protocols
11. Single-cell vs single nucleus RNA-Seq processing
12. Lessons learned & challenges
13. Q&A

PRESENTERS

Judit Jané-Valbuena, Ph.D, Scientific Manager, Klarman Cell Observatory, Broad Institute

Sébastien Vigneau, Ph.D. Head of the Innovation Lab, Center for Cancer Genomics, Dana-Farber Cancer Institute

Michal Slyper, Ph.D. Research Scientist, Klarman Cell Observatory, Broad Institute