College of Veterinary Medicine > Research > Bioinformatics > Single cell guide

Single cell guide

Step 1: Single Cell Suspension Preparation

1. Sample Collection

Artistic drawing of tissue with a piece of it being cut away so that it can be analyzed.

2. Tissue mincing

Artistic drawing of tissue on a petri dish.

3. Tissue preparation

Artistic drawing of petri dish with tissue cut in small pieces.

4. Mechanical and enzymatic dissociation

Artistic drawing of vial with liquid and tissue (about 1/2 full), and an orange cap.

5. Sample prepared

Sample in vial in piece of equipment.

6. Single Cell Suspension

Vial with suspesion of the tissue, still half full, but now the liquid is the color of the tissue.

Spending time researching and learning how to create a quality single cell suspension BEFORE generating cDNA libraries with the 10x Chromium platform is essential.

A general rule of thumb is that if you are proficient in flow cytometry cell preparations you are prepared for the 10X Chromium step.

Preparation of single cell suspension from tissue samples or culture systems is the most important step in single-cell-RNA-seq.

Screen grab from video.
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Step 2: Microfluidics with 10x Chromium controller

The single-cell preparation from tissue will need to be run through a micro-fluidics machine that is made by 10x Genomics. This is done by Lisette Maddison.

Device being loaded by a person who is wearing a protective glove.
A Chromium Controller from 10X Genomics.

Step 3: cDNA library Preparation and Illumina Sequencing

GEMd mRNA is then turned into cDNA and amplified in order to prepare for the next step which is Illumina sequencing.

After successful GEM preparations cDNA libraries will be constructed, QCd, and amplified at the Molecular Biology and Genomics Core. Contact Derek Pouchnik (pooch@wsu.edu) at the Molecular Biology and Genomics Core to plan and perform this part of the process.

Laboratory for Biotechnology and Bioanalysis Molecular Biology and Genomics Core
Nexteq2000 machine
The Illumina NextSeq 2000 DNA sequencer is shown in a lab in the Biotechnology Life Sciences Building in Washington State University’s College of Veterinary Medicine on Friday, Sept. 15, 2023, in Pullman. (College of Veterinary Medicine/Ted S. Warren)

Step 4: Sequence Alignment

Person looking at server

Sequence files called ‘FASTQs” will be generated by the Illumina sequencer that can be processed by using WSU’s high performance computer cluster called ‘Kamiak’, which has the 10x specific sequence aligner called ‘Cellranger’. Contact kamiak.support@wsu.edu to establish a user account and to receive instructions on how to align your FASTQs.

Step 5: Data Analysis

Once you have completed sequence alignment with CellRanger on Kamiak you can contact VIS to setup a cloud based server that will be ready for you to analyze the output files using Seurat on R.

Sequence file sample

Veterinary information systems

College of Veterinary Medicine Datahub: This is the CVM datahub that hosts webtools that are part of manuscript.

Technical Assistance or Service Inquiries:
509-335-0101 or help@vetmed.wsu.edu