Nature Methods Application Notes e-UPDATE: 10 February 2015

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10 February 2015  Application Notes bring you the latest information about innovative tools and technologies and their applications in the lab. We hope that you will find this service useful and informative and encourage you to sign up for future Updates to ensure that you never miss one!

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FEATURED APPLICATION NOTE Fast and Convenient 5-hydroxymethylcytosine Enrichment Workflow for Next-Generation Sequencing www.thermofisher.com > The Scientific™ EpiJET™ 5-hmC Enrichment Kit is highly specific for different DNA samples containing 5-hydroxymethylcytosine (5-hmC), an extensively studied DNA epigenetic modification. This tool, when combined with next-generation sequencing, offers new ways of analyzing 5-hmC at the genomic level. The data show that this tool can be used for both locus-specific and whole genome–specific analysis, yielding 5-hmC distribution patterns across different genomes. PDF

		Reporter Assays and More: Applications of NanoLuc® Luciferase www.promega.com > NanoLuc® Luciferase brings exciting new possibilities and improvements to luminescence applications, including protein stability monitoring, detection of protein interactions using BRET, and in vivo imaging. This article highlights several recent papers that illustrate the use of NanoLuc® Technology as a sensitive reporter for challenging applications beyond those of classic bioluminescence reporter assays. PDF

		QuantSeq 3′ mRNA sequencing for RNA quantification www.lexogen.com > QuantSeq provides an easy protocol to generate highly strand-specific next-generation sequencing (NGS) libraries close to the 3′ end of polyadenylated RNAs within 4.5 h. Only one fragment per transcript is generated, directly linking the number of reads mapping to a gene to its expression. QuantSeq reduces data analysis time and enables a higher level of multiplexing per run. QuantSeq is the RNA sample preparation method for accurate and affordable gene expression measurement. PDF

		Approaching Single-Cell Sequencing by Understanding NGS Library Complexity and Bias www.swiftbiosci.com > Demands are growing on genomics to deliver higher quality sequencing data from samples with less input quantity. As the number of genomic equivalents decreases at lower input amounts, library bias and library complexity increasingly affect data quality. Less biased, more complex libraries result in more even and complete coverage, resulting in better sequencing efficiency and reduced costs. This application note describes the sequence coverage performance and preservation of molecular complexity of next generation sequencing (NGS) libraries generated from human and microbial genomic DNA using the Accel-NGS™ 2S DNA Library Kit for whole-genome sequencing (WGS) on the Illumina® platform. PDF

		Using PrimeScript™ Reverse Transcriptase for Quantification of shRNA Knockdown of a Low Abundance Target www.clontech.com > PrimeScript Reverse Transcriptase is a high efficiency enzyme that is ideal for preparing cDNA for the analysis of low abundance transcripts. In this experiment, Cdc14A mRNA, which is expressed at low levels, was quantified in mouse embryonic fibroblast (MEF) cells that had been transduced with either a control shRNA or a Cdc14A-specific shRNA. The PrimeScript RT Reagent Kit (Perfect Real Time) (Cat. #RR037A)and a reverse transcriptase from another supplier (Company I) were used to generate cDNA templates for measuring Cdc14A expression by real-time PCR using SYBR® Green detection. PDF

		Chromatrap® 96: a new solid-state platform for high-throughput ChIP www.chromatrap.com > Chromatrap® 96 (C96) is a high-throughput analysis platform that profiles up to 96 transcription factors and epigenetic modifications simultaneously in less than 1 d. It enables sensitive, selective and reproducible target amplification with excellent signal-to-noise ratios, even from samples as small as 0.1 μg. Compatible with automated handling, C96 allows simultaneous investigation of parallel epigenetic landscapes, offering unprecedented assay flexibility and speed. PDF

		Optimized Library Prep for Cell-Free DNA from Human Plasma www.biooscientific.com > Researchers and clinicians are increasingly interested in using Next Generation Sequencing (NGS) analysis of cell-free DNA (cfDNA) found in plasma (the cell-free fraction of anticoagulated blood) for biomarker discovery and diagnostic applications. Two areas of specific interest are non-invasive prenatal diagnostics (1-6) and monitoring efficacy of treatment in cancer patients (7-20). Many recent studies have shown the feasibility of detecting fetal aneuploidies such as Chromosome 21 trisomy (the cause of Down's Syndrome) by shotgun sequencing of DNA-Seq libraries produced from cell-free DNA isolated from maternal blood. Other aneuploidies including trisomies of Chromosomes 13 and 18 have also been detected (3). This approach is attractive since it avoids the risk of miscarriage associated with invasive tests (amniocentesis and chorionic villi sampling) and may offer cost benefits for prenatal diagnosis. PDF

 

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