Customer Developed Protocol

Tissue dissociation and single cell preparationof breast cancer patient-derived xenografts
Contributed by:

Ioannis Ragoussis, Ph. D. and Morag Park, Ph. D.

CUSTOMER DEVELOPED PROTOCOL
Tissue dissociation and single cell preparation of breast cancer patient-derived xenografts

Contributor Research Profile (Ioannis Ragoussis)
The Ragoussis lab works on Technical Developments including the Single Cell Genomics Lab of MUGQIC andMutation Detection Technologies with focus on the genomic and transcriptomic analysis of single cells. Specifically,
his group focuses on 4 main areas: 1) Single cell genomics to study tumor heterogeneity, tumor microenvironment,
and circulating tumor cells, 2) Methodologies for the highly sensitive detection of both somatic as well as germline
mutations and different cancer types, 3) The development of long read single molecule sequencing based
methodologies and diagnostic tests, and 4) High throughput whole genome genotyping using Affymetrix Axiom
technology as part of Canada’s Longitudinal Study of Aging.
Link to the lab: http://www.mcgillgenomecentre.org/ioannis-ragoussis/

Contributor Research Profile (Morag Park)
The Park lab aims to identify new signal transduction pathways that are important for the development of humancancers and how these can be targeted with drug therapies. This is a complex question that requires a fullunderstanding of how signals are integrated in normal cells and how these signals become altered in tumor cells, inthe context of other genetic alterations. Specifically, Park’s research focuses on 1) Studying mechanisms ofactivation and cell transformation by receptor tyrosine kinases, 2) Investigating the role of Gab Family Signal
Amplifiers in Epithelial Morphogenesis and tumorigenesis, 3) Identifying signals regulating epithelial cell motility
and invasion in breast cancer
Link to the lab: https://www.mcgill.ca/biochemistry/about-us/department/faculty-members/parkReferenceThis protocol was used in:
Savage et al. A Targetable EGFR-Dependent Tumor-Initiating Program in Breast Cancer, Cell Reports,
2017, 21(5):1140-49
https://www.ncbi.nlm.nih.gov/pubmed/29091754

10x Genomics Products
10x Chromium™ Single Cell Gene Expression Solution - https://www.10xgenomics.com/single-cell/Notices
THIS PROTOCOL IS NOT SUPPORTED BY 10X GENOMICSThis Customer Developed Protocol is provided for general information only and is not directly supported,
endorsed, or certified by 10x Genomics. 10x Genomics gives no warranties and makes no claims about theprovided protocol.
For questions or to share protocol adjustments, please visit the 10x Community at community.10xgenomics.com

Customer Developed Protocol

COMMUNITY.10XGENOMICS.COM

CUSTOMER DEVELOPED PROTOCOL
Tissue dissociation and single cell preparation of breast cancer patient-derived xenografts

Required Buffers and Reagents
1. MACS Octo Dissociator with Heater (Miltenyi)
2. Mouse Cell Depletion Kit (Miltenyi)
3. PBS with 0.04% BSA

ProtocolSingle-cell suspensions were generated by mechanical and enzymatic dissociation using the gentle MACS Octo
dissociator with heaters (Miltenyi), according to manufacturer’s protocol. Murine stromal cells were removed
using a Mouse Cell Depletion Kit (Miltenyi), according to manufacturer’s protocol.
Mouse-depleted single-cell suspensions were washed three times in PBS with 0.04% BSA. An aliquot of cells wasused for LIVE/DEAD viability testing (Thermo Fisher Scientific). Single-cell libraries were generated using theGemCode Single-Cell Instrument, the Chromium™ Single Cell 3' Library & Gel Bead Kit v2 and Chromium™ Single
Cell A Chip Kit (10x Genomics), according to the manufacturer’s protocol.

THIS PROTOCOL IS NOT SUPPORTED BY 10X GENOMICSThis 10x Community Customer Developed Protocol is provided for general information only andis not directly supported, endorsed, or certified by 10x Genomics. 10x Genomics gives no warranties and makes
no claims about the provided protocol.
For questions or to share protocol adjustments, please visit the 10x Community at community.10xgenomics.com

Customer Developed Protocol

COMMUNITY.10XGENOMICS.COM