Single cell guide

Step 1: Single Cell Suspension Preparation

1. Sample Collection

2. Tissue mincing

Tissue Mincing

3. Tissue preparation

Tissue Preperation

4. Mechanical and enzymatic dissociation

5. Sample prepared

Sample prepared

6. Single Cell Suspension

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.

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.

Lisette Maddison, an Assistant Research Professor with the Center for Reproductive Biology in the College of Veterinary Medicine at Washington State University, works in her lab with a Chromium Controller from 10X Genomics device on Thursday, Jan. 19, 2023, in Pullman. (College of Veterinary Medicine/Ted S. Warren)

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 ( at the Molecular Biology and Genomics Core to plan and perform this part of the process.

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 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:
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