Nature Methods Application Notes e-UPDATE: 8 October 2013

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8 OCTOBER 2013  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 High-content analysis of three-dimensional tumor spheroids: investigating signaling pathways using small hairpin RNA www.3dbiomatrix.com > www.ttplabtech.com > http://www.gnf.org > In the past, creating three-dimensional (3D) tumor spheroids that were suitable for high-throughput screening (HTS) was a difficult and often expensive process. We describe how to couple easy, controllable 3D spheroid formation in Perfecta3D® Hanging Drop Plates with the acumen® eX3 high-content imager for rapid, multicolor, whole-well quantification. This process provides researchers with a highly efficient method to achieve physiologically relevant tumor models in an HTS format. PDF

		An Integrated Solution to Simplify Library Preparation and Multiplexing for NimbleGen Sequence Capture http://www.biooscientific.org > Targeted sequencing is an important tool in analyzing disease or exome mutations. In this study, we describe how Bioo Scientific's NEXTflex™ DNA Pre-Capture Combo Library construction kits were used in conjunction with NimbleGen Sequence Capture technology to obtain high coverage comparative genomic data from a panel of human HapMap DNA samples. We identified 96-98% of known HapMap SNPs in the NimbleGen SeqCap EZ Exome v3.0 (64 Mb) and SeqCap EZ Design — Comprehensive Cancer Design (3.9 Mb) probes with a high percentage of reads mapping to the targeted regions. This study illustrates both the ability of Bioo Scientific's NEXTflex library construction kit to produce high-quality material for NGS, and the robust performance of the NimbleGen Sequence Capture technology. PDF

		Optimized method for rapid protein electroblotting www.thermoscientific.com > Protein electroblotting is a common method for transferring proteins out of a polyacrylamide gel onto a membrane for subsequent western blotting. With a high-current power supply, such as that found in the Thermo Scientific™ Pierce™ G2 Fast Blotter, and a high–ionic strength transfer buffer, protein transfer from nitrocellulose or polyvinylidene difluoride (PVDF) membranes can be achieved in 5–10 m, even with proteins as large as 300 kDa. PDF

		Faster, cleaner bisulfite conversion that yields more intact DNA for downstream analysis www.promega.com > Performing DNA purification, bisulfite conversion and amplification are crucial steps in DNA methylation analysis, especially when working with difficult sample types like formalin-fixed paraffin-embedded (FFPE) tissue. The MethylEdge™ Bisulfite Conversion System provides a rapid, efficient method for bisulfite conversion with minimal DNA fragmentation in less than 2 h. Coupled with purification chemistries optimized for FFPE tissue and flexible, robust amplification technologies for detection, the MethylEdge™ Bisulfite Conversion System delivers high-quality converted DNA for downstream analysis. PDF

		EpiGnome™ Methyl-Seq Kit: a novel post–bisulfite conversion library prep method for methylation analysis www.epibio.com > Epigenomics is increasingly becoming an important field of research, and the ability to detect and quantify DNA methylation accurately is now critical for numerous fields of study, including disease biology and gene expression. The differential reactivities of methylated and nonmethylated cytosines in DNA with sodium bisulfite forms the basis for their identification in the genome by sequencing. Current whole-genome bisulfite sequencing methods require substantial amounts of starting DNA to compensate for the loss due to bisulfite-mediated DNA degradation. To address issues with sample preparation, we have developed the EpiGnome™ Methyl-Seq Kit, which utilizes a novel pre-library bisulfite conversion scheme to prepare whole-genome bisulfite sequencing libraries with no sample loss and with only 50 ng of input genomic DNA. PDF

		A transposable approach to RNA-seq from total RNA www.epibio.com > The rapid progress of RNA-seq technology requires the development of simple, versatile tools that competently reflect RNA levels in the cell. We have developed the TotalScript™ RNA-Seq Kit, a new, transposome-based directional library prep method suitable for a variety of RNA sample types and a range of input. TotalScript libraries exhibit high complexity, ~97–99% directionality and low rRNA contamination. The method is scalable and robust, allowing input amounts of ~100 pg of poly(A)+ RNA or ~5 ng of total RNA. PDF

		Endotoxin-free protein production—ClearColi™ technology www.lucigen.com > A new Escherichia coli strain with a genetically modified lipopolysaccharide (LPS) molecule has been created that enables protein expression without the endotoxin. Proteins expressed and purified from ClearColi™ BL21(DE3) cells will not trigger LPS-related immune response in human cells, making them ideal for immunogenicity, toxicity and cytokine assays. PDF

 

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