Induced pluripotent stem cells (iPSCs) are an essential tool for modeling how causal genetic variants impact cellular function in disease, as well as an emerging source of tissue for regenerative medicine. The preparation of somatic cells, their reprogramming and the subsequent verification of iPSC pluripotency are laborious, manual processes limiting the scale and reproducibility of this technology. Here we describe a modular, robotic platform for iPSC reprogramming enabling automated, high-throughput conversion of skin biopsies into iPSCs and differentiated cells with minimal manual intervention. We demonstrate that automated reprogramming and the pooled selection of polyclonal pluripotent cells results in high-quality, stable iPSCs. These lines display less line-to-line variation than either manually produced lines or lines produced through automation followed by single-colony subcloning. The robotic platform we describe will enable the application of iPSCs to population-scale biomedical problems including the study of complex genetic diseases and the development of personalized medicines.
Summary The Phylogenetic Analysis with Space/Time models (PHAST) package is a widely used software package for comparative genomics that has been freely available for download since 2002. Here, we introduce a web interface (phastWeb) that makes it possible to use two of the most popular programs in PHAST, phastCons and phyloP, without downloading and installing the PHAST software. This interface allows users to upload a sequence alignment and either upload a corresponding phylogeny or have one estimated from the alignment. After processing, users can visualize alignments and conservation scores as genome browser tracks and download estimated tree models and raw scores for further analysis. Altogether, this resource makes key features of the PHAST package conveniently available to a broad audience. Availability and implementation PhastWeb is freely available on the web at http://compgen.cshl.edu/phastweb/. The website provides instructions as well as examples.
Background The concentrations of distinct types of RNA in cells result from a dynamic equilibrium between RNA synthesis and decay. Despite the critical importance of RNA decay rates, current approaches for measuring them are generally labor-intensive, limited in sensitivity, and/or disruptive to normal cellular processes. Here, we introduce a simple method for estimating relative RNA half-lives that is based on two standard and widely available high-throughput assays: Precision Run-On sequencing (PRO-seq) and RNA sequencing (RNA-seq). Results Our method treats PRO-seq as a measure of transcription rate and RNA-seq as a measure of RNA concentration, and estimates the rate of RNA decay required for a steady-state equilibrium. We show that this approach can be used to assay relative RNA half-lives genome-wide, with good accuracy and sensitivity for both coding and noncoding transcription units. Using a structural equation model (SEM), we test several features of transcription units, nearby DNA sequences, and nearby epigenomic marks for associations with RNA stability after controlling for their effects on transcription. We find that RNA splicing-related features are positively correlated with RNA stability, whereas features related to miRNA binding and DNA methylation are negatively correlated with RNA stability. Furthermore, we find that a measure based on U1 binding and polyadenylation sites distinguishes between unstable noncoding and stable coding transcripts but is not predictive of relative stability within the mRNA or lincRNA classes. We also identify several histone modifications that are associated with RNA stability. Conclusion We introduce an approach for estimating the relative half-lives of individual RNAs. Together, our estimation method and systematic analysis shed light on the pervasive impacts of RNA stability on cellular RNA concentrations.
The rate at which RNA molecules are degraded is a key determinant of cellular RNA concentrations, yet current approaches for measuring RNA half-lives are generally laborintensive, limited in sensitivity, and/or disruptive to normal cellular processes. Here we introduce a simple method for estimating relative RNA half-lives that is based on two standard and widely available high-throughput assays: Precision Run-On and sequencing (PRO-seq) and RNA sequencing (RNA-seq). Our method treats PRO-seq as a measure of transcription rate and RNAseq as a measure of RNA concentration, and estimates the rate of RNA degradation required for a steady-state equilibrium. We show that this approach can be used to assay relative RNA halflives genome-wide, with reasonable accuracy and good sensitivity for both coding and noncoding transcription units. Using a structural equation model (SEM), we test several features of transcription units, nearby DNA sequences, and nearby epigenomic marks for associations with RNA stability after controlling for their effects on transcription. We find that RNA splicing-related features, including intron length, are positively correlated with RNA stability, whereas features related to miRNA binding, DNA methylation, and G+C-richness are negatively correlated with RNA stability. Furthermore, we find that a measure of predicted stability based on U1 binding sites and polyadenylation sites distinguishes between unstable noncoding and stable coding transcripts but is not predictive of relative stability within the mRNA or lincRNA classes. We also identify several histone modifications that are associated with RNA stability after controlling for their correlations with transcription. Together, our estimation method and systematic analysis shed light on the pervasive impacts of RNA stability on cellular RNA concentrations.
Increased global connectivity has catalyzed technological development in almost all industries, in part through the facilitation of novel collaborative structures. Notably, open innovation and crowd-sourcing-of expertise and/ or funding-has tremendous potential to increase the effi ciency with which biomedical ecosystems interact to deliver safe, effi cacious and affordable therapies to patients. Consequently, such practices offer tremendous potential in advancing development of cellular therapies.In this vein, the CASMI Translational Stem Cell Consortium (CTSCC) was formed to unite global thought-leaders, producing academically rigorous and commercially practicable solutions to a range of challenges in pluripotent stem cell translation. Critically, the CTSCC research agenda is defi ned through continuous consultation with its international funding and research partners.Herein, initial fi ndings for all research focus areas are presented to inform global product development strategies, and to stimulate continued industry interaction around biomanufacturing, strategic partnerships, standards, regulation and intellectual property and clinical adoption.
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