Renan Escalante-Chong | Search bioRxiv

bioRxiv2016

Cell freezing protocol optimized for ATAC-Seq on motor neurons derived from human induced pluripotent stem cells

In recent years, the assay for transposase-accessible chromatin using sequencing (ATAC-Seq) has become a fundamental tool of epigenomic research. However, it has proven difficult to perform this technique on frozen samples because freezing cells before extracting nuclei impairs nuclear integrity and alters chromatin structure. We describe a protocol for freezing cells that is compatible with ATAC-Seq, producing results that compare well with those generated from fresh cells. We found that while flash-frozen samples are not suitable for ATAC-Seq, the assay is successful with slow-cooled cryopreserved samples. Using this method, we were able to isolate high quality, intact nuclei, and we verified that epigenetic results from fresh and cryopreserved samples agree quantitatively. We developed our protocol on a disease-relevant cell type, namely motor neurons differentiated from induced pluripotent stem cells from a patient affected by spinal muscular atrophy.

Genomics↗

bioRxiv2014

Natural Variation in Preparation for Nutrient Depletion Reveals a Cost-Benefit Tradeoff

Maximizing growth and survival in the face of a complex, time-varying environment is a common problem for single-celled organisms in the wild. When offered two different sugars as carbon sources, microorganisms first consume the preferred sugar, then undergo a transient growth delay, the \"diauxic lag\", while inducing genes to metabolize the less preferred sugar. This delay is commonly assumed to be an inevitable consequence of selection to maximize use of the preferred sugar. Contrary to this view, we found that many natural isolates of Saccharomyces cerevisiae display short or non-existent diauxic lags when grown in mixtures of glucose (preferred) and galactose. These strains induce galactose-utilization (GAL) genes hours before glucose exhaustion, thereby \"preparing\" for the transition from glucose to galactose metabolism. The extent of preparation varies across strains, and seems to be determined by the steady-state response of GAL genes to mixtures of glucose and galactose rather than by induction kinetics. Although early GAL induction gives strains a competitive advantage once glucose runs out, it comes at a cost while glucose is still present. Costs and benefits correlate with the degree of preparation: strains with higher expression of GAL genes prior to glucose exhaustion experience a larger upfront growth cost but also a shorter diauxic lag. Our results show that classical diauxic growth is only one extreme on a continuum of growth strategies constrained by a cost-benefit tradeoff. This type of continuum is likely to be common in nature, as similar tradeoffs can arise whenever cells evolve to use mixtures of nutrients.

Systems Biology↗