Concurrent analysis of genome and transcriptome in one single cell.

Thus far, multiple techniques for single cell analysis have been developed, yet we lack a relatively simple tool to assess DNA and RNA from the same cell at whole-transcriptome and whole-genome depths. Here we present an updated method for physical separation of cytoplasmic RNA from the nuclei, which allows for simultaneous studies of DNA and RNA from the same single cell. The method consists of three steps-(1) immobilization of a single cell on solid substrate, (2) hypotonic lysis of immobilized single cell, and (3) separation of cytosol containing aqueous phase and immobilized nucleus. We found that DNA and RNA extracted from single cell using our approach is suitable for downstream sequencing-based applications. We demonstrated that the coverage of transcriptome and genome sequencing data obtained after DNA/RNA separation is similar to that observed without separation. We also showed that the separation procedure does not create any noticeable bias in observed mutational load or mutation spectra. Thus, our method can serve as a tool for simultaneous complex analysis of the genome and transcriptome, providing necessary information on the relationship between somatic mutations and the regulation of gene expression.

Negative selection allows human primary fibroblasts to tolerate high somatic mutation loads induced by N-ethyl-N-nitrosourea.

Single-cell sequencing has shown that thousands of mutations accumulate with age in most human tissues. While there is ample evidence that some mutations can clonally amplify and lead to disease, the total burden of mutations a cell tolerates without functional decline remains unknown. Here we addressed this question by exposing human primary fibroblasts to multiple, low doses of N-ethyl-N-nitrosourea (ENU) and analyzed somatic mutation burden using single-cell whole genome sequencing. The results indicate that individual cells can sustain ∼60,000 single-nucleotide variants (SNVs) with only a slight adverse effect on growth rate. We provide evidence that such high levels of mutations are only tolerated through negative selection against variants in gene coding regions, and in sequences associated with genetic pathways for maintaining basic cellular function and growth. Since most tissues in adults are non-dividing, these results suggest that somatic mutations in the absence of negative selection may have functionally adverse effects.

Concurrent analysis of genome and transcriptome in one single cell.

Thus far, multiple techniques for single cell analysis have been developed, yet we lack a relatively simple tool to assess DNA and RNA from the same cell at whole-transcriptome and whole-genome depths. Here we present an updated method for physical separation of cytoplasmic RNA from the nuclei, which allows for simultaneous studies of DNA and RNA from the same single cell. The method consists of three steps - 1) immobilization of a single cell on solid substrate, 2) hypotonic lysis of immobilized single cell, and 3) separation of cytosol containing aqueous phase and immobilized nucleus. We found that DNA and RNA extracted from single cell using our approach is suitable for downstream sequencing-based applications. We demonstrated that the coverage of transcriptome and genome sequencing data obtained after DNA/RNA separation is similar to that observed without separation. We also showed that the separation procedure does not create any noticeable bias in observed mutational load or mutation spectra. Thus, our method can serve as a tool for simultaneous complex analysis of the genome and transcriptome, providing necessary information on the relationship between somatic mutations and the regulation of gene expression.

Single-molecule, quantitative detection of low-abundance somatic mutations by high-throughput sequencing.

Postzygotic somatic mutations have been found associated with human disease, including diseases other than cancer. Most information on somatic mutations has come from studying clonally amplified mutant cells, based on a growth advantage or genetic drift. However, almost all somatic mutations are unique for each cell, and the quantitative analysis of these low-abundance mutations in normal tissues remains a major challenge in biology. Here, we introduce single-molecule mutation sequencing (SMM-seq), a novel approach for quantitative identification of point mutations in normal cells and tissues.

Aging Triggers H3K27 Trimethylation Hoarding in the Chromatin of Nothobranchius furzeri Skeletal Muscle.

Aging associates with progressive loss of skeletal muscle function, sometimes leading to sarcopenia, a process characterized by impaired mobility and weakening of muscle strength. Since aging associates with profound epigenetic changes, epigenetic landscape alteration analysis in the skeletal muscle promises to highlight molecular mechanisms of age-associated alteration in skeletal muscle. This study was conducted exploiting the short-lived turquoise killifish Nothobranchius furzeri (Nfu), a relatively new model for aging studies. The epigenetic analysis suggested a less accessible and more condensed chromatin in old Nfu skeletal muscle. Specifically, an accumulation of heterochromatin regions was observed as a consequence of increased levels of H3K27me3, HP1α, polycomb complex subunits, and senescence-associated heterochromatic foci (SAHFs). Consistently, euchromatin histone marks, including H3K9ac, were significantly reduced. In this context, integrated bioinformatics analysis of RNASeq and ChIPSeq, related to skeletal muscle of Nfu at different ages, revealed a down-modulation of genes involved in cell cycle, differentiation, and DNA repair and an up-regulation of inflammation and senescence genes. Undoubtedly, more studies are needed to disclose the detailed mechanisms; however, our approach enlightened unprecedented features of Nfu skeletal muscle aging, potentially associated with swimming impairment and reduced mobility typical of old Nfu.

Age-dependent increase of oxidative stress regulates microRNA-29 family preserving cardiac health.

The short-lived turquoise killifish Nothobranchius furzeri (Nfu) is a valid model for aging studies. Here, we investigated its age-associated cardiac function. We observed oxidative stress accumulation and an engagement of microRNAs (miRNAs) in the aging heart. MiRNA-sequencing of 5 week (young), 12-21 week (adult) and 28-40 week (old) Nfu hearts revealed 23 up-regulated and 18 down-regulated miRNAs with age. MiR-29 family turned out as one of the most up-regulated miRNAs during aging. MiR-29 family increase induces a decrease of known targets like collagens and DNA methyl transferases (DNMTs) paralleled by 5´methyl-cytosine (5mC) level decrease. To further investigate miR-29 family role in the fish heart we generated a transgenic zebrafish model where miR-29 was knocked-down. In this model we found significant morphological and functional cardiac alterations and an impairment of oxygen dependent pathways by transcriptome analysis leading to hypoxic marker up-regulation. To get insights the possible hypoxic regulation of miR-29 family, we exposed human cardiac fibroblasts to 1% O2 levels. In hypoxic condition we found miR-29 down-modulation responsible for the accumulation of collagens and 5mC. Overall, our data suggest that miR-29 family up-regulation might represent an endogenous mechanism aimed at ameliorating the age-dependent cardiac damage leading to hypertrophy and fibrosis.