Optimized high-purity isolation of developing new macronuclei in <i>Tetrahymena thermophila</i> enables epigenetic analyses

Abstract The de novo establishment of epigenetic modifications plays a pivotal role in gene expression, maintaining genome stability, and cell differentiation. Tetrahymena thermophila provides an attractive model for studying de novo establishment of epigenetic modifications, as its developing new macronucleus (MAC) undergoes extensive epigenetic reprogramming during the sexual process of conjugation. Nevertheless, the coexistence of the new MACs with the parental MACs and new micronuclei (MICs) within the same cell, as well as parental MACs and MICs in non-mating cells within the same culture, has hindered detailed studies. To address this issue, we developed an optimized purification strategy including initial enrichment of new MACs by differential centrifugation, followed by further purification using fluorescence-activated cell sorting (FACS). With this two-step approach, we ultimately obtained new MACs with over 99% purity. This method will provide a crucial foundation for leveraging the new MAC to dissect the de novo establishment of epigenetic marks and their potential regulatory roles.