The Challenges of Genome-Wide Studies in a Unicellular Eukaryote With Two Nuclear Genomes.

We present here methods to study a eukaryotic microorganism with two nuclear genomes, both originating from the same zygotic genome. Paramecium, like other ciliates, is characterized by nuclear dimorphism, which is the presence of two types of nuclei with distinct organization and functions in the same cytoplasm. The two diploid germline micronuclei (MIC) undergo meiosis and fertilization to transmit the genetic information across sexual generations. The highly polyploid somatic macronucleus (MAC) contains a reduced version of the genome optimized for gene expression. Reproducible programmed DNA elimination of about 30% of the complexity of the 100Mb MIC genome occurs during development of the MAC along with endoreplication to 800 copies. Large regions that contain transposable elements and other repeats are eliminated, and short single copy remnants of transposable elements, which often interrupt coding sequences, are precisely excised to restore functional open reading frames. Genome-wide studies of this process require access to MIC DNA which has long been impossible. The breakthrough with respect to this technical obstacle came with development of a MIC purification protocol involving a critical step of flow cytometry to sort nuclei representing only 0.5% of total genomic DNA. Here, we provide a step-by-step protocol and important tips for purifying nuclei, and present the methods developed for downstream analysis of NGS data.