Customer Developed Protocol

Isolation of single cell suspensions fromepidermis
Contributed by:Samuel Lukowski, Ph.D.

CUSTOMER DEVELOPED PROTOCOL
Isolation of single cell suspensions from epidermis

Contributor Research Profile
The Powell lab develops and applies computational and statistical genomics approaches to investigate the genetic
control of genome regulation and its role in contributing to the susceptibility to human disease. Specifically, ourresearch involves the use of large-scale transcriptomic and DNA sequence data from both bulk tissues and single
cells, focusing on understanding the genetic mechanisms by which heritable variants contribute to diseasesusceptibility at a cellular level, and ultimately achieve therapeutic and diagnostic outcomes.
Link to the lab: https://imb.uq.edu.au/single-cell-and-computational-genomics

Reference
This protocol was used in:
Lukowski et al. Detection of HPV E7 transcription at single-cell resolution in epidermis., bioRxiv doi:
https://doi.org/10.1101/252858
https://www.biorxiv.org/content/early/2018/01/24/252858.full.pdf+html

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

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CUSTOMER DEVELOPED PROTOCOL
Isolation of single cell suspensions from epidermis

Required Buffers and Reagents
1. 2.5 µg/µL Dispase II (Roche)
2. 1 μg/μL Collagenase D (Roche)
3. 0.2 μg/μL DNase (Roche)
4. 0.7 μm filter (BD Falcon)
5. 7-AAD (0.25 μg, eBioscience)
6. Rat anti-mouse CD19/CD32 (5 µg, clone 93, eBioscience)
7. APC-conjugated rat anti-mouse CD45.2 antibodies (0.5 µg, Clone 104, Biolegend)
8. PBS + 2% serum + 2 mM EDTA

Protocol
Tissue collection and dissociation
Skin biopsies were split into dorsal and ventral parts and incubated with Dispase II (Roche) for 1 h at 37°C.
Epidermis and dermis were separated with closed forceps. The epidermis was further homogenized anddigested with 1 µg/µL of collagenase D (Roche) and 0.2 µg/µL of DNase, (Roche) for 1 h at 37°C. Digested
samples were passed through a 0.7 µm filter (BD Falcon) to generate a single cell suspension for staining andsorting.

Cell sorting
Flow cytometry of single cell suspensions of digested epidermis samples were incubated with Fc Block (5 µg, rat
anti-mouse CD19/CD32, clone 93, eBioscience) diluted in PBS for 30 min on ice. Samples were subsequently
incubated with APC-conjugated rat anti-mouse CD45.2 antibodies (0.5 µg, Clone 104, Biolegend) diluted in PBS +2% serum + 2 mM EDTA for 30 min on ice. Prior to sorting, cells were labelled with 7-AAD (0.25 µg,eBioscience). Live CD45- cells (7-AAD- and APC- or PE-Cy7-) were sorted using the BD ARIA Fusion sorter at 12 psiwith a 100 µm nozzle. ~300,000-500,000 events/cells were collected per sample.

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