Single-cell mapping of homocysteine-induced perturbations in avian embryogenesis using LMO and BD rhapsody transcriptomics.

Single-cell RNA sequencing enables the study of cellular heterogeneity and the effects of environmental perturbations across complex tissues. However, subtle biological responses can be obscured by experimental noise, sample-specific processing, and computational integration. Here, we combined lipid-modified oligonucleotide cell tagging with the BD Rhapsody platform to multiplex early embryonic chick cells exposed to different homocysteine conditions. This species-agnostic approach enabled robust sample barcoding, simultaneous processing of treatment conditions, and recovery of high-quality single-cell transcriptomes from heterogeneous embryonic tail tissues. By comparing non-integrated and integrated analyses, we found that integration preserved broad cell identities but could mask cluster-specific transcriptional signatures in sensitive populations. Using the non-integrated multiplexed dataset, we identified homocysteine-associated transcriptional changes in neuromesodermal progenitor-derived populations and linked them to posterior mesodermal and axis elongation defects. This workflow provides an adaptable strategy for detecting subtle treatment effects in non-traditional developmental models.