A single-nucleus census of immune and non-immune cell types for the major immune organ systems of chicken

In the avian host, comprehensively cataloging immune cell types, their transcriptome profiles, and varying molecular responses to pathogen challenges are necessary steps toward a better understanding of the interplay between genetics and disease resilience. We present a first nuclei atlas of immune cell types derived from the three main immune organs of layer chickens, including spleen, bursa, and thymus. In bursa we also present, an accounting of cell type activation with the bacterial toxin lipopolysaccharide (LPS). Our analysis includes 36,370 total nuclei and 16, 12, and 12 transcriptionally distinct clusters for spleen, bursa, and thymus, respectively. We discover nuclei molecular profiles that uniquely distinguish states of the transcriptome within cell type that could serve as new means to characterize avian immune subtypes. We further subcluster refined immune cell type classifications, specifically highlighting the transcriptomic diversity of B and T cell subtypes. In the bursa, inferred intercellular communication and signaling pathway enrichment analyses across immune and non-immune cell types demonstrate the unappreciated complexity of the B cell repertoire in a model mimicking systemic bacterial infection. This census of all cell types in both primary and one major secondary avian immune organ system, although preliminary, provides a first review of how nuclei transcribe numerous genes, known and unknown, a critical prerequisite for the study avian immunogenetics by cell type. We used the Singulator2 instrument per manufacturer’s protocols (S2 Genomics; Livermore, CA) with storage buffers containing 0.4U/ul RNase inhibitor (source), prior to microfluidic encapsulation on the 10X Chromium instrument (10x Genomics®, Pleasanton, CA) to nanoliter-scale Gel bead-in-EMulsions (GEMs). Single-nuclei libraries were generated using the GemCode Single-Cell Instrument and Single Cell 3′ Library and Gel Bead Kit v3 and Chip Kit (10x Genomics®, Pleasanton, CA) according to the manufacturer’s protocol. Before sequencing, every library was analyzed on a Bioanalyzer high sensitivity chip to ensure the expected cDNA fragment size distribution was achieved. The appropriate number of individually barcoded GEM libraries were pooled and sequenced on a NovaSeq 6000 instrument (Illumina) with 2×150bp length using these sequencing parameters: 26 bp read 1 – 8 bp index 1 (i7) – 98 bp read 2 with 200 cycles.