Single-cell transcriptome analysis of channel catfish whole spleen and IgM-positive splenic fractions provides insight into the fish immunome from an aquaculture-relevant species

Authors: ALDERSEY, JOHANNA - Orise Fellow; Lange, Miles; Beck, Benjamin; Abernathy, Jason

Submitted to: Advances in Genome Biology and Technology
Publication Type: Abstract Only
Publication Acceptance Date: 2/22/2024
Publication Date: 4/15/2024
Citation: Aldersey, J., Lange, M.D., Beck, B.H., Abernathy, J.W. 2024. Single-cell transcriptome analysis of channel catfish whole spleen and IgM-positive splenic fractions provides insight into the fish immunome from an aquaculture-relevant species. Advances in Genome Biology and Technology[abstract].

Interpretive Summary:

Technical Abstract: The catfish industry is the largest sector of U.S. aquaculture production. Additionally, channel catfish, Ictalurus punctatus, is an important model for studying teleost fish immunity. Given its role in food production, the catfish immune response to industry-relevant pathogens has been extensively studied and has provided crucial information on innate and adaptive immune function during disease progression. To further examine the channel catfish immune system, we performed single-cell RNA sequencing on both whole spleens (n=3) and IgM-positive splenic B-cells (n=3). Spleen cell suspensions were prepared by passing tissues through a cell sieve. For IgM-positive B-cells, spleen lymphocytes were isolated and then labeled with a recombinant monoclonal antibody that is reactive to catfish IgM and sorted via flow cytometry. Single-cell RNAseq libraries were then prepared using the 10X Genomics Chromium X with the Next GEM Single Cell 3’ Reagents and sequenced on an Illumina NovaSeq X Plus. Each demultiplexed sample was aligned to the CoCo_2.0 channel catfish reference assembly, filtered, and counted to generate feature-barcode matrices. From whole spleen samples, outputs were analyzed both individually (SP1, SP2, and SP3) and as a normalized aggregate. SP1, SP2, and SP3 generated 198,304,083-422,617,575 reads from an estimated 5,140-11,463 cells with approximately 25,594-41,874 reads/cell. The reads aligned to 22,234-23,144 genes and 59–80% of reads had a valid barcode and mapped confidently to the transcriptome. The median number of genes found per cell was 554-733. The aggregated cell data had 688,285,722 reads from an estimated 24,351 number of cells and approximately 28,265 reads/cell. 73% of reads had a valid barcode and mapped confidently to the transcriptome and a median of 664 genes were found per cell. K-means analysis on individual samples found nine groups with significantly altered gene expression. Initially, two major clusters were identified including erythrocytes (2,516–4,934 cells) and B-cells (405–2235 cells). Additional gene expression profiles within whole spleen samples as well as IgM-sorted data analytics will be presented and discussed. These data will serve as the foundation for a spleen cell atlas of this primary immune tissue in fish and allow for profiling of B-cell subsets to a high-resolution.