ATAC-seq of low-input and cryopreserved primordial germ cells reveals functional enhancers

Dynamic changes in chromatin accessibility at cis-regulatory elements underlie cell fate transitions during development. Primordial germ cells (PGCs) represent a rare population whose chromatin dynamics remain poorly understood compared to known epigenetic landscapes. Here, we utilized the chicken PGC model to bridge this gap, leveraging its capacity for in vitro expansion and in vivo colonization. We adapted the ATAC-seq workflow to obtain reproducible accessible chromatin region (ACR) profiles from as few as 200 cells, even after cryopreservation. Integrative analysis identified over 10,000 PGC-specific ACRs, many absent from somatic tissues, and revealed inherent Tn5 transposase sequence biases in the chicken genome. To validate these ACRs, we established an in vitro PGC differentiation system and utilized in vivo embryonic transplantation. Reporter assays confirmed enhancer activities in cultured PGCs, while transcriptome integration associated these ACRs with genes expressed at embryonic day 2.5. In vivo transplantation demonstrated that these enhancers exhibited early stage-specific activity, becoming silenced upon gonadal settlement. Our results provide a practical strategy for identifying functional regulatory elements from minimal starting material, facilitating the study of chromatin dynamics in rare cell populations.