SUMMARY Massively parallel single-cell RNA sequencing can precisely resolve cellular diversity in a high-throughput manner at low cost, but unbiased isolation of intact single cells from complex tissues, such as adult mammalian brains, is challenging. Here, we integrate sucrose-gradient assisted purification of nuclei with droplet microfluidics to develop a highly scalable single-nucleus RNA-Seq approach (sNucDrop-Seq), which is free of enzymatic dissociation and nuclei sorting. By profiling ~18,000 nuclei isolated from cortical tissues of adult mice, we demonstrate that sNucDrop-Seq not only accurately reveals neuronal and non-neuronal subtype composition with high sensitivity, but also enables in-depth analysis of transient transcriptional states driven by neuronal activity, at single-cell resolution, in vivo.

Here we present APOBEC-Coupled Epigenetic Sequencing (ACE-Seq), a bisulfite-free method for localizing 5-hydroxymethylcytosine (5hmC) at single-base resolution with low DNA input. The method builds upon the observation that AID/APOBEC family DNA deaminase enzymes can potently discriminate between cytosine modification states, and exploits the non-destructive nature of enzymatic, rather than chemical, deamination. ACE-Seq yields high-confidence 5hmC profiles with at least 1000-fold less DNA input than conventional methods. Applying ACE-Seq to generate a base-resolution map of 5hmC in tissue-derived cortical excitatory neurons, we find that 5hmC is almost entirely confined to CG dinucleotides. The map permits cytosine, 5-methylcytosine (5mC) and 5hmC to be parsed and reveals genomic features that diverge from global patterns, including enhancers and imprinting control regions with high and low 5hmC/5mC ratios, respectively. Enzymatic deamination overcomes many challenges posed by bisulfite-based methods and expands the scope of epigenome profiling to include scarce samples and open new lines of inquiry regarding the role of cytosine modifications in genome biology.

Molecular mechanisms of wound healing have been extensively characterized, providing a better understanding of the processes involved in wound repair and offering advances in treatment methods. Both spatial and temporal investigations of injury biomarkers have helped to pinpoint significant time points and locations during the recovery process, which may be vital in managing the injury and making the appropriate diagnosis. This study addresses spatial and temporal differences of phosphoproteins found in skeletal muscle tissue following a traumatic femur fracture, which were further compared to co-localized cytokine responses. In particular, several proteins (Akt, ERK, c-Jun, CREB, JNK, MEK1, and p38) and post-translational phosphorylations (p-Akt, p-c-Jun, p-CREB, p-ERK1/2, p-MEK1, p-p38, p-GSK3α/β, p-HSP27, p-p70S6K, and p-STAT3) associated with inflammation, new tissue formation, and remodeling were found to exhibit significant spatial and temporal differences in response to the traumatic injury. Quadratic discriminant analysis of all measured responses, including cytokine concentrations from previously published findings, was used to classify temporal and spatial observations at high predictive rates, further confirming that distinct spatiotemporal distributions for total protein, phosphorylation signaling, and cytokine (IL-1α, IL-1ß, IL2, IL6, TNF-α, and MIP-1α) responses exist. Finally, phosphoprotein measurements were found to be significantly correlated to cytokine concentrations, suggesting coordinated intracellular and extracellular activity during crucial periods of repair. This study represents a first attempt to monitor and assess integrated changes in extracellular and intracellular signaling in response to a traumatic injury in muscle tissues, which may provide a framework for future research to improve both our understanding of wounds and their treatment options.

Thousands of gallons of industrial chemicals, crude 4-methylcyclohexanemethanol (MCHM) and propylene glycol phenyl ether (PPh), leaked from industrial tanks into the Elk River in Charleston, West Virginia, USA, on January 9, 2014. A considerable number of people were reported to exhibit symptoms of chemical exposure and an estimated 300,000 residents were advised not to use or drink tap water. At the time of the spill, the existing toxicological data of the chemicals were limited for a full evaluation of the health risks, resulting in concern among those in the impacted regions. In this preliminary study, we assessed cell viability and plasma membrane degradation following a 24-h exposure to varying concentrations (0-1000 μM) of the two compounds, alone and in combination. Evaluation of different cell lines, HEK-293 (kidney), HepG2 (liver), H9c2 (heart), and GT1-7 (brain), provided insight regarding altered cellular responses in varying organ systems. Single exposure to MCHM or PPh did not affect cell viability, except at doses much higher than the estimated exposure levels. Certain co-exposures significantly reduced metabolic activity and increased plasma membrane degradation in GT1-7, HepG2, and H9c2 cells. These findings highlight the importance of examining co-exposures to fully understand the potential toxic effects.