Single-cell RNA seq mapping of chicken leukocytes

The immune system is a complex infrastructure where many cells interact with each other and perform duties depending on their type and function. When using traditional immunological methods in studying non-traditional model organisms, such as birds, challenges arise. These are often associated with a lack of knowledge surrounding the organism in question—particularly, the expected leukocytes, their cell-specific marker genes, and associated reagents. Single-cell transcriptomics allows us to study the immune system at the level of each singular cell and create a profile of each cell present in a sample without as much prior knowledge of the organism. This project aimed to investigate the possibility of using single-cell transcriptomics as an alternative to traditional methods in avian immunology and using this as a basis for further research into avian medicine. The study was performed by sequencing the mRNA in approximately twenty thousand individual chicken blood cells from 4 healthy adult birds, performing unsupervised clustering of the cells, and attempting to annotate clusters based on expression profiles. Most of this study has been performed using the R-based software Seurat and 10 x genomics software Cell Ranger. Resulting putative cell types include expected populations such as several different T-cells, B-cells, Monocytes, thrombocytes, red blood cells, and cells in various stages of the cell life cycle. After computational analysis, the number of cells per cell type corresponds to laboratory analysis of the cell types performed prior to sequencing. This indicates that the in-silico annotation of putative cell types is consistent with the actual cell types in the samples This study of chicken leukocytes highlights the possibility of the usage of single-cell transcriptomics within non-traditional model organism immunology. It shows that using modern single-cell sequencing and existing software, sequencing-based characterisation of immune cells is possible and could prove a robust option in immunology study cases where traditional methods are limited. Overall design: Blood samples from chickens were purified to remove RBCs and Thrombocytes using gradient centrifugation on Ficoll and immunomagnetic cell separation respectively and analyzed using scRNA-seq. Read counting was performed using Cell Ranger count and data analysis was performed using Seurat.