Project description:A new cost-effective ribosome profiling technique complements the functional genome annotation and reveals a key role of genic 3’ untranslated region in plant translatomic variation
Project description:A new cost-effective ribosome profiling technique complements the functional genome annotation and reveals a key role of genic 3’ untranslated region in plant translatomic variation
Project description:A new cost-effective ribosome profiling technique complements the functional genome annotation and reveals a key role of genic 3’ untranslated region in plant translatomic variation
Project description:A new cost-effective ribosome profiling technique complements the functional genome annotation and reveals a key role of genic 3’ untranslated region in plant translatomic variation
Project description:Ribosome footprint profiling is a high throughput sequencing based technique that provides detailed and global views of translation in living cells. An essential part of this technology is removal of unwanted, normally abundant, ribosomal RNA sequences in order to save on sequencing costs. The most effective commercial solution (Ribo-Zero) has been discontinued and a number of new, experimentally distinct commercial applications have emerged on the market. Here we evaluated several commercially available alternatives designed for RNA-seq of human samples and find them unsuitable for ribosome footprint profiling. We instead recommend the use of custom-designed biotinylated oligos, which were widely used in early ribosome profiling studies. Importantly, we warn that depletion solutions based on targeted nuclease cleavage significantly perturb the high-resolution information that can be derived from the data, and thus do not recommend their use for any applications that require precise determination of the ends of RNA fragments.
Project description:We developed a novel approach, J-binding protein 1 sequencing (JBP1-seq), that combines the benefits of an improved recombinant JBP1 protein, Nextera-based library construction, and nextgeneration sequencing (NGS) for genome-wide profiling of 5-hydroxymethylcytosine (5hmC). Compared with the original JBP1, this new recombinant JBP1 was biotinylatedin vivo and conjugated to magnetic beads via biotin-streptavidin interactions. These modifications allowed a more efficient and consistent pull-down of β-glucosyl-5-hydroxymethylcytosine (β-glu-5hmC), and sequence-ready libraries can be generated within 4.5 hours from DNA inputs as low as 50 ng. 5hmC enrichment of human brain DNA using the new JBP1 resulted in over 25,000 peaks called, which is significantly higher than the 4,003 peaks enriched using the old JBP1. Comparison of the technical duplicates and validations with other platforms indicated the results are reproducible and reliable. Thus, JBP1-seq provides a fast, efficient, cost-effective method for accurate 5hmC genome-wide profiling. An improvement of JBP1-Seq
Project description:<p>Droplet-based single-cell RNA-seq has emerged as a powerful technique for massively parallel cellular profiling. While these approaches offer the exciting promise to deconvolute cellular heterogeneity in diseased tissues, the lack of cost-effective, reliable, and user-friendly instrumentation has hindered widespread adoption of droplet microfluidic techniques. To address this, we have developed a microfluidic control instrument that can be easily assembled from 3D printed parts and commercially available components costing approximately $575. We adapted this instrument for massively parallel scRNA-seq and deployed it in a clinical environment to perform single-cell transcriptome profiling of disaggregated synovial tissue from 5 rheumatoid arthritis patients. We sequenced 20,387 single cells from synovectomies, revealing 13 transcriptomically distinct clusters. These encompass a comprehensive and unbiased characterization of the autoimmune infiltrate, including inflammatory T and NK subsets that contribute to disease biology. Additionally, we identified fibroblast subpopulations that are demarcated via THY1 (CD90) and CD55 expression. Further experiments confirm that these represent synovial fibroblasts residing within the synovial intimal lining and subintimal lining, respectively, each under the influence of differing microenvironments. We envision that this instrument will have broad utility in basic and clinical settings, enabling low-cost and routine application of microfluidic techniques, and in particular single-cell transcriptome profiling.</p> <p>Reprinted from [Stephenson et al., Nature Communications, 2018], with permission from the Nature Publishing Group.</p>
Project description:We established a novel and cost-effective procedure to isolate extracellular vesicles from bovine milk via salting-out. We aimed to obtain the profiling of the small RNAs in these EVs as an important characterization in EV research.
Project description:A new high-density oligonucleotide array of the human transcriptome (GG-H array) has been developed for high-throughput and cost-effective analyses in clinical studies. This array allows comprehensive examination of gene expression and genome-wide identification of alternative splicing, as well as detection of coding SNPs and non-coding transcripts. The GG-H array was validated using samples from multiple independent preparations of human liver and muscle, and compared with results obtained from mRNA sequencing analysis. The GG-H array is highly reproducible in estimating gene and exon abundance, and is sensitive in detecting expression changes and alternative splicing. This array has been implemented in a multi-center clinical program and has generated high quality, reproducible data. When current cost, as well as sample and time requirements for sequencing are considered in the context of a required throughput of hundreds of samples per week for a clinical trial, the array provides a high-throughput and cost effective platform for clinical genomic studies. Examination exon/gene expression of liver and muscle in quadraplicates using both the array technology and RNA-Seq
Project description:A new high-density oligonucleotide array of the human transcriptome (GG-H array) has been developed for high-throughput and cost-effective analyses in clinical studies. This array allows comprehensive examination of gene expression and genome-wide identification of alternative splicing, as well as detection of coding SNPs and non-coding transcripts. The GG-H array was validated using samples from multiple independent preparations of human liver and muscle, and compared with results obtained from mRNA sequencing analysis. The GG-H array is highly reproducible in estimating gene and exon abundance, and is sensitive in detecting expression changes and alternative splicing. This array has been implemented in a multi-center clinical program and has generated high quality, reproducible data. When current cost, as well as sample and time requirements for sequencing are considered in the context of a required throughput of hundreds of samples per week for a clinical trial, the array provides a high-throughput and cost effective platform for clinical genomic studies. Examination exon/gene expression of liver and muscle in quadraplicate using both the array technology and RNA-Seq
