Integrity of chromatin and replicating DNA in nuclei released from fission yeast by semi-automated grinding in liquid nitrogen

Abstract

Abstract Background Studies of nuclear function in many organisms, especially those with tough cell walls, are limited by lack of availability of simple, economical methods for large-scale preparation of clean, undamaged nuclei. Findings Here we present a useful method for nuclear isolation from the important model organism, the fission yeast, Schizosaccharomyces pombe. To preserve in vivo molecular configurations, we flash-froze the yeast cells in liquid nitrogen. Then we broke their tough cell walls, without damaging their nuclei, by grinding in a precision-controlled motorized mortar-and-pestle apparatus. The cryo-ground cells were resuspended and thawed in a buffer designed to preserve nuclear morphology, and the nuclei were enriched by differential centrifugation. The washed nuclei were free from contaminating nucleases and have proven well-suited as starting material for genome-wide chromatin analysis and for preparation of fragile DNA replication intermediates. Conclusions We have developed a simple, reproducible, economical procedure for large-scale preparation of endogenous-nuclease-free, morphologically intact nuclei from fission yeast. With appropriate modifications, this procedure may well prove useful for isolation of nuclei from other organisms with, or without, tough cell walls.