Search

The generation of quality data from a single-nucleus profiling experiment requires nuclei to be isolated from tissues in a gentle and efficient manner. Nuclei isolation must be carefully optimized across tissue types to preserve nuclear architecture, prevent nucleic acid degradation, and remove unwanted contaminants. Here, we present an optimized workflow for generating a single-nucleus suspension from ocular tissues of the embryonic chicken that is compatible with various downstream workflows. The described protocol enables the rapid isolation of a high yield of aggregate-free nuclei from the embryonic chicken eye without compromising nucleic acid integrity, and the nuclei suspension is compatible with single-nucleus RNA and ATAC sequencing. We detail several stopping points, either via cryopreservation or fixation, to enhance workflow adaptability. Further, we provide a guide through multiple QC points and demonstrate proof-of-principle using two commercially available kits. Finally, we demonstrate that existing in silico genotyping methods can be adopted to computationally derive biological replicates from a single pool of chicken nuclei, greatly reducing the cost of biological replication and allowing researchers to consider sex as a variable during analysis. Together, this tutorial represents a cost-effective, simple, and effective approach to single-nucleus profiling of embryonic chicken eye tissues and is likely to be easily modified to be compatible with similar tissue types., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Ocular lens development entails epithelial to fiber cell differentiation, defects in which cause congenital cataracts. We report the first single-cell multiomic atlas of lens development, leveraging snRNA-seq, snATAC-seq and CUT&RUN-seq to discover previously unreported mechanisms of cell fate determination and cataract-linked regulatory networks. A comprehensive profile of cis- and trans-regulatory interactions, including for the cataract-linked transcription factor MAF, is established across a temporal trajectory of fiber cell differentiation. Furthermore, we identify an epigenetic paradigm of cellular differentiation, defined by progressive loss of the H3K27 methylation writer Polycomb repressive complex 2 (PRC2). PRC2 localizes to heterochromatin domains across master-regulator transcription factor gene bodies, suggesting it safeguards epithelial cell fate. Moreover, we demonstrate that FGF hyper-stimulation in vivo leads to MAF network activation and the emergence of novel lens cell states. Collectively, these data depict a comprehensive portrait of lens fiber cell differentiation, while defining regulatory effectors of cell identity and cataract formation., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)