An improved protocol for small RNA library construction using High Definition adapters

AbstractNext generation sequencing of small RNA (sRNA) libraries is widely used for studying sRNAs in various biological systems. However, cDNA libraries of sRNAs are biased for molecules that are ligated to adapters more or less efficiently than other molecules. One approach to reduce this ligation bias is to use a pool of adapters instead of a single adapter sequence, which allows many sRNAs to be ligated efficiently. We previously developed High Definition (HD) adapters for the Illumina sequencing platform, which contain degenerate nucleotides at the ligating ends of the adapters. However, the current commercial kits produced a large amount of 5’ adapter – 3’ adapter ligation product without the cDNA insert when HD adapters were used to replace the kit adapters. Here, we report a protocol to generate sRNA libraries using HD adapters with greatly reduced proportion of adapter-adapter products due to the degradation of nonligated 3’ adapters. The libraries can be completed within two days and can be used for various biological and clinical samples. As examples for using this protocol, we constructed sRNA libraries using total RNA extracted from cultured mammalian cells and plant leaf tissue.

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Applications of Flow Cytometry to Clinical Microbiology

Clinical Microbiology Reviews ◽

10.1128/cmr.13.2.167 ◽

2000 ◽

Vol 13 (2) ◽

pp. 167-195 ◽

Cited By ~ 67

Author(s):

Alberto Álvarez-Barrientos ◽

Javier Arroyo ◽

Rafael Cantón ◽

César Nombela ◽

Miguel Sánchez-Pérez

Keyword(s):

Flow Cytometry ◽

Clinical Microbiology ◽

Ex Vivo ◽

Analytical Techniques ◽

Clinical Samples ◽

Clinical Microbiology Laboratory ◽

Commercial Kits ◽

Antimicrobial Treatments ◽

Near Future

SUMMARY Classical microbiology techniques are relatively slow in comparison to other analytical techniques, in many cases due to the need to culture the microorganisms. Furthermore, classical approaches are difficult with unculturable microorganisms. More recently, the emergence of molecular biology techniques, particularly those on antibodies and nucleic acid probes combined with amplification techniques, has provided speediness and specificity to microbiological diagnosis. Flow cytometry (FCM) allows single- or multiple-microbe detection in clinical samples in an easy, reliable, and fast way. Microbes can be identified on the basis of their peculiar cytometric parameters or by means of certain fluorochromes that can be used either independently or bound to specific antibodies or oligonucleotides. FCM has permitted the development of quantitative procedures to assess antimicrobial susceptibility and drug cytotoxicity in a rapid, accurate, and highly reproducible way. Furthermore, this technique allows the monitoring of in vitro antimicrobial activity and of antimicrobial treatments ex vivo. The most outstanding contribution of FCM is the possibility of detecting the presence of heterogeneous populations with different responses to antimicrobial treatments. Despite these advantages, the application of FCM in clinical microbiology is not yet widespread, probably due to the lack of access to flow cytometers or the lack of knowledge about the potential of this technique. One of the goals of this review is to attempt to mitigate this latter circumstance. We are convinced that in the near future, the availability of commercial kits should increase the use of this technique in the clinical microbiology laboratory.

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Small RNA Profiling by Next-Generation Sequencing Using High-Definition Adapters

Methods in Molecular Biology - MicroRNA Detection and Target Identification ◽

10.1007/978-1-4939-6866-4_4 ◽

2017 ◽

pp. 45-57 ◽

Cited By ~ 2

Author(s):

Martina Billmeier ◽

Ping Xu

Keyword(s):

Next Generation Sequencing ◽

Small Rna ◽

Next Generation ◽

High Definition ◽

Rna Profiling ◽

Generation Sequencing

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Functional genomics approach to hypoxia signaling

Journal of Applied Physiology ◽

10.1152/japplphysiol.00836.2003 ◽

2004 ◽

Vol 96 (2) ◽

pp. 765-773 ◽

Cited By ~ 46

Author(s):

Karen A. Seta ◽

David E. Millhorn

Keyword(s):

Cell Survival ◽

Mammalian Cells ◽

Cdna Libraries ◽

Vascular Endothelial ◽

Pheochromocytoma Cells ◽

Adaptive Mechanisms ◽

Endothelial Growth Factor ◽

Microarray Analyses ◽

Expressed Sequence ◽

Sustained Hypoxia

Mammalian cells require a constant supply of oxygen to maintain energy balance, and sustained hypoxia can result in cell death. It is therefore not surprising that sophisticated adaptive mechanisms have evolved that enhance cell survival during hypoxia. During the past few years, there have been a growing number of reports on hypoxia-induced transcription of specific genes. In this review, we describe a unique experimental approach that utilizes focused cDNA libraries coupled to microarray analyses to identify hypoxia-responsive signal transduction pathways and genes that confer the hypoxia-tolerant phenotype. We have used the subtractive suppression hybridization (SSH) method to create a cDNA library enriched in hypoxia-regulated genes in oxygen-sensing pheochromocytoma cells and have used this library to create microarrays that allow us to examine hundreds of genes at a time. This library contains over 300 genes and expressed sequence tags upregulated by hypoxia, including tyrosine hydroxylase, vascular endothelial growth factor, and junB. Hypoxic regulation of these and other genes in the library has been confirmed by microarray, Northern blot, and real-time PCR analyses. Coupling focused SSH libraries with microarray analyses allows one to specifically study genes relevant to a phenotype of interest while reducing much of the biological noise associated with these types of studies. When used in conjunction with high-throughput, dye-based assays for cell survival and apoptosis, this approach offers a rapid method for discovering validated therapeutic targets for the treatment of cardiovascular disease, stroke, and tumors.

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Small RNA Profile in Moso Bamboo Root and Leaf Obtained by High Definition Adapters

PLoS ONE ◽

10.1371/journal.pone.0103590 ◽

2014 ◽

Vol 9 (7) ◽

pp. e103590 ◽

Cited By ~ 10

Author(s):

Ping Xu ◽

Irina Mohorianu ◽

Li Yang ◽

Hansheng Zhao ◽

Zhimin Gao ◽

...

Keyword(s):

Small Rna ◽

Moso Bamboo ◽

High Definition

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The Absence of C-5 DNA Methylation in Leishmania donovani Allows DNA Enrichment from Complex Samples

Microorganisms ◽

10.3390/microorganisms8081252 ◽

2020 ◽

Vol 8 (8) ◽

pp. 1252

Author(s):

Bart Cuypers ◽

Franck Dumetz ◽

Pieter Meysman ◽

Kris Laukens ◽

Géraldine De Muylder ◽

...

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Leishmania Donovani ◽

Clinical Samples ◽

Knock Out ◽

The Status ◽

Genome Bisulfite Sequencing ◽

Genomic Scale ◽

Commercial Kits ◽

Eukaryotic Organisms

Cytosine C5 methylation is an important epigenetic control mechanism in a wide array of eukaryotic organisms and generally carried out by proteins of the C-5 DNA methyltransferase family (DNMTs). In several protozoans, the status of this mechanism remains elusive, such as in Leishmania, the causative agent of the disease leishmaniasis in humans and a wide array of vertebrate animals. In this work, we showed that the Leishmania donovani genome contains a C-5 DNA methyltransferase (DNMT) from the DNMT6 subfamily, whose function is still unclear, and verified its expression at the RNA level. We created viable overexpressor and knock-out lines of this enzyme and characterized their genome-wide methylation patterns using whole-genome bisulfite sequencing, together with promastigote and amastigote control lines. Interestingly, despite the DNMT6 presence, we found that methylation levels were equal to or lower than 0.0003% at CpG sites, 0.0005% at CHG sites, and 0.0126% at CHH sites at the genomic scale. As none of the methylated sites were retained after manual verification, we conclude that there is no evidence for DNA methylation in this species. We demonstrated that this difference in DNA methylation between the parasite (no detectable DNA methylation) and the vertebrate host (DNA methylation) allowed enrichment of parasite vs. host DNA using methyl-CpG-binding domain columns, readily available in commercial kits. As such, we depleted methylated DNA from mixes of Leishmania promastigote and amastigote DNA with human DNA, resulting in average Leishmania:human enrichments from 62× up to 263×. These results open a promising avenue for unmethylated DNA enrichment as a pre-enrichment step before sequencing Leishmania clinical samples.

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Comparison of the utility of five commercial kits for extraction of DNA from Aspergillus fumigatus spores.

Acta Biochimica Polonica ◽

10.18388/abp.2010_2445 ◽

2010 ◽

Vol 57 (4) ◽

Cited By ~ 9

Author(s):

Urszula Nawrot ◽

Katarzyna Wlodarczyk ◽

Magdalena Wrobel ◽

Anita Wasik ◽

Tadeusz Dobosz

Keyword(s):

Dna Extraction ◽

Enzymatic Digestion ◽

Cell Disruption ◽

Clinical Samples ◽

Rrna Gene ◽

28S Rrna ◽

Bacterial Dna ◽

Blood Samples ◽

Dna Yield ◽

Commercial Kits

The aim of this study was to compare the efficiency of DNA extraction from water as well as from blood samples spiked with A. fumigatus spores, using selected commercial kits. Extraction of DNA according to manufacturer's protocols was preceded by blood cells lysis and disruption of fungal cells by enzymatic digestion or bead beating. The efficiency of DNA extraction was measured by PCR using Aspergillus-specific primers and SYBR Green I dye or TaqMan probes targeting 28S rRNA gene. All methods allowed the detection of Aspergillus at the lowest tested density of water suspensions of spores (10¹ cells/ml). The highest DNA yield was obtained using the ZR Fungal/Bacterial DNA kit, YeastStar Genomic DNA kit, and QIAamp DNA Mini kit with mechanical cell disruption. The ZR Fungal/Bacterial DNA and YeastStar kits showed the highest sensitivity in examination of blood samples spiked with Aspergillus (100 % for the detection of 10² spores and 75 % for 10¹ spores). Recently, the enzymatic method ceased to be recommended for examination of blood samples for Aspergillus, thus ZR Fungal/Bacterial DNA kit and QIAamp DNA Mini kit with mechanical cell disruption could be used for extraction of Aspergillus DNA from clinical samples.

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Identification of peroxisomal proteins by using M13 phage protein VI phage display: molecular evidence that mammalian peroxisomes contain a 2,4-dienoyl-CoA reductase

Biochemical Journal ◽

10.1042/bj3400561 ◽

1999 ◽

Vol 340 (2) ◽

pp. 561-568 ◽

Cited By ~ 22

Author(s):

Marc FRANSEN ◽

Paul P. VAN VELDHOVEN ◽

Suresh SUBRAMANI

Keyword(s):

Mammalian Cells ◽

Unsaturated Fatty Acids ◽

Urate Oxidase ◽

Cdna Libraries ◽

Molecular Evidence ◽

Cdna Clones ◽

Novel Proteins ◽

M13 Phage ◽

Coa Reductase ◽

Protein Vi

To elucidate unknown mammalian peroxisomal enzymes and functions, we subjected M13 phage expressing fusions between the gene encoding protein VI and a rat liver cDNA library to an immunoaffinity selection process in vitro (biopanning) with the use of antibodies raised against peroxisomal subfractions. In an initial series of biopanning experiments, four different cDNA clones were obtained. These cDNA species encoded two previously identified peroxisomal enzymes, catalase and urate oxidase, and two novel proteins that contained a C-terminal peroxisomal targeting signal (PTS1). A primary structure analysis of these novel proteins revealed that one, ending in the tripeptide AKL, is homologous to the yeast peroxisomal 2,4-dienoyl-CoA reductase (EC 1.3.1.34; DCR), an enzyme required for the degradation of unsaturated fatty acids, and that the other, ending in the tripeptide SRL, is a putative member of the short-chain dehydrogenase/reductase (SDR) family, with three isoforms. Green fluorescent protein (GFP) fusions encoding GFP-DCR-AKL, GFP-DCR, GFP-SDR-SRL and GFP-SDR were expressed in mammalian cells. The analysis of the subcellular location of the recombinant fusion proteins confirmed the peroxisomal localization of GFP-DCR-AKL and GFP-SDR-SRL, as well as the functionality of the PTS1. That the AKL protein is indeed an NADPH-dependent DCR was demonstrated by showing DCR activity of the bacterially expressed protein. These results demonstrate at the molecular level that mammalian peroxisomes do indeed contain a DCR. In addition, the results presented here indicate that the protein VI display system is suitable for the isolation of rare cDNA clones from cDNA libraries and that this technology facilitates the identification of novel peroxisomal proteins.

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