Gel-free multiplexed reduced representation bisulfite sequencing for large-scale DNA methylation profiling.

Sequencing-based approaches have led to new insights about DNA methylation. While many different techniques for genome-scale mapping of DNA methylation have been employed, throughput has been a key limitation for most. To further facilitate the mapping of DNA methylation, we describe a protocol for gel-free multiplexed reduced representation bisulfite sequencing (mRRBS) that reduces the workload dramatically and enables processing of 96 or more samples per week. mRRBS achieves similar CpG coverage to the original RRBS protocol, while the higher throughput and lower cost make it better suited for large-scale DNA methylation mapping studies, including cohorts of cancer samples.

Universality of protein reentrant condensation in solution induced by multivalent metal ions.

The effective interactions and phase behavior of protein solutions under strong electrostatic coupling conditions are difficult to understand due to the complex charge pattern and irregular geometry of protein surfaces. This distinguishes them from related systems such as DNA or conventional colloids. In this work, we discuss the question of universality of the reentrant condensation (RC) of proteins in solution induced by multivalent counterions, i.e., redissolution on adding further salts after phase separation, as recently discovered (Zhang et al., Phys Rev Lett 2008; 101:148101). The discussion is based on a systematic investigation of five different proteins with different charge patterns and five different multivalent counterions. Zeta potential measurements confirm the effective charge inversion of proteins in the reentrant regime via binding of multivalent counterions, which is supported by Monte Carlo simulations. Charge inversion by trivalent cations requires an overall negative net charge of the protein. Statistical analysis of a representative set of protein sequences reveals that, in theory, this effect could be possible for about half of all proteins. Our results can be exploited for the control of the phase behavior of proteins, in particular facilitating protein crystallization.