Illumina mate-paired DNA sequencing-library preparation using Cre-Lox recombination

Standard Illumina mate-paired libraries are constructed from 3- to 5-kb DNA fragments by a blunt-end circularization. Sequencing reads that pass through the junction of the two joined ends of a 3-5-kb DNA fragment are not easy to identify and pose problems during mapping and de novo assembly. Longer read lengths increase the possibility that a read will cross the junction. To solve this problem, we developed a mate-paired protocol for use with Illumina sequencing technology that uses Cre-Lox recombination instead of blunt end circularization. In this method, a LoxP sequence is incorporated at the junction site. This sequence allows screening reads for junctions without using a reference genome. Junction reads can be trimmed or split at the junction. Moreover, the location of the LoxP sequence in the reads distinguishes mate-paired reads from spurious paired-end reads. We tested this new method by preparing and sequencing a mate-paired library with an insert size of 3 kb from Saccharomyces cerevisiae. We present an analysis of the library quality statistics and a new bio-informatics tool called DeLoxer that can be used to analyze an IlluminaCre-Lox mate-paired data set. We also demonstrate how the resulting data significantly improves a de novo assembly of the S. cerevisiae genome

Whole genome transcript profiling from fingerstick blood samples: a comparison and feasibility study

<p>Abstract</p> <p>Background</p> <p>Whole genome gene expression profiling has revolutionized research in the past decade especially with the advent of microarrays. Recently, there have been significant improvements in whole blood RNA isolation techniques which, through stabilization of RNA at the time of sample collection, avoid bias and artifacts introduced during sample handling. Despite these improvements, current human whole blood RNA stabilization/isolation kits are limited by the requirement of a venous blood sample of at least 2.5 mL. While fingerstick blood collection has been used for many different assays, there has yet to be a kit developed to isolate high quality RNA for use in gene expression studies from such small human samples. The clinical and field testing advantages of obtaining reliable and reproducible gene expression data from a fingerstick are many; it is less invasive, time saving, more mobile, and eliminates the need of a trained phlebotomist. Furthermore, this method could also be employed in small animal studies, i.e. mice, where larger sample collections often require sacrificing the animal. In this study, we offer a rapid and simple method to extract sufficient amounts of high quality total RNA from approximately 70 μl of whole blood collected via a fingerstick using a modified protocol of the commercially available Qiagen PAXgene RNA Blood Kit.</p> <p>Results</p> <p>From two sets of fingerstick collections, about 70 uL whole blood collected via finger lancet and capillary tube, we recovered an average of 252.6 ng total RNA with an average RIN of 9.3. The post-amplification yields for 50 ng of total RNA averaged at 7.0 ug cDNA. The cDNA hybridized to Affymetrix HG-U133 Plus 2.0 GeneChips had an average % Present call of 52.5%. Both fingerstick collections were highly correlated with r²values ranging from 0.94 to 0.97. Similarly both fingerstick collections were highly correlated to the venous collection with r²values ranging from 0.88 to 0.96 for fingerstick collection 1 and 0.94 to 0.96 for fingerstick collection 2.</p> <p>Conclusions</p> <p>Our comparisons of RNA quality and gene expression data of the fingerstick method with traditionally processed sample workflows demonstrate excellent RNA quality from the capillary collection as well as very high correlations of gene expression data.</p

Differential transcriptome analysis of glandular and filamentous trichomes in Artemisia annua

Background: The medicinal plant Artemisia annua is covered with filamentous trichomes and glandular, artemisinin producing trichomes. A high artemisinin supply is needed at a reduced cost for treating malaria. Artemisinin production in bioreactors can be facilitated if a better insight is obtained in the biosynthesis of artemisinin and other metabolites. Therefore, metabolic activities of glandular and filamentous trichomes were investigated at the transcriptome level. Results: By laser pressure catapulting, glandular and filamentous trichomes as well as apical and sub-apical cells from glandular trichomes were collected and their transcriptome was sequenced using Illumina RNA-Seq. A de novo transcriptome was assembled (Trinity) and studied with a differential expression analysis (edgeR). A comparison of the transcriptome from glandular and filamentous trichomes shows that MEP, MVA, most terpene and lipid biosynthesis pathways are significantly upregulated in glandular trichomes. Conversely, some transcripts coding for specific sesquiterpenoid and triterpenoid enzymes such as 8-epi-cedrol synthase and an uncharacterized oxidosqualene cyclase were significantly upregulated in filamentous trichomes. All known artemisinin biosynthesis genes are upregulated in glandular trichomes and were detected in both the apical and sub-apical cells of the glandular trichomes. No significant differential expression could be observed between the apical and sub-apical cells. Conclusions: Our results underscore the vast metabolic capacities of A. annua glandular trichomes but nonetheless point to the existence of specific terpene metabolic pathways in the filamentous trichomes. Candidate genes that might be involved in artemisinin biosynthesis are proposed based on their putative function and their differential expression level

Glycogene Expression in Conjunctiva of Patients with Dry Eye: Downregulation of Notch Signaling

purpose. Glycoconjugates regulate a variety of biological events in mucosal surfaces, such as differentiation of postmitotic epithelial cells and maintenance of the wet-surfaced phenotype. This study aimed to identify the repertoire of genes (glycogenes) involved in biosynthesis of glycoconjugates in conjunctiva of normal subjects and patients with dry eye. methods. RNA from conjunctival impression cytology samples was amplified and hybridized to a custom-designed glycogene microarray. Intensity data were converted to expression values and analyzed by ANOVA. Microarray data for selected Notch glycogenes were confirmed by quantitative real-time PCR. Notch receptors and ligands were immunolocalized on conjunctival biopsies by confocal microscopy. results. By microarray, 424 glycogenes were identified in normal conjunctival epithelium; galectins, glycosyltransferases, mucins, Notch signaling molecules, and proteoglycans were among the most highly expressed. In dry eye, 46 glycogenes were significantly downregulated, including five members of the Notch signaling pathway (Notch1, Notch 2, Notch 3, Jagged1, Delta1), four Wnt signaling molecules (Wnt4, -5A, Frizzled6, -7), and three heparan sulfate glycotransferases (HS2ST1, HS3ST6, EXTL2). Only interferon-induced transmembrane protein 1 was upregulated. By real-time PCR, expression ratios of Notch1, Notch 3, and Jagged1 in dry eye were 0.43, 0.56, and 0.50, respectively, compared to controls (P < 0.05). Notch1, Notch3, and Jagged1 were immunolocalized throughout the conjunctival epithelium, whereas Notch2 and Delta1 were distributed apically. conclusions. This study revealed the differential glycogene expression profiles in normal subjects and patients with dry eye. Downregulation of Notch signaling in dry eye may result in abnormal differentiation of the conjunctival epithelium and have implications in the pathogenesis of the disease

The long noncoding RNA THRIL regulates TNFalpha expression through its interaction with hnRNPL

Thousands of large intergenic noncoding RNAs (lincRNAs) have been identified in the mammalian genome, many of which have important roles in regulating a variety of biological processes. Here, we used a custom microarray to identify lincRNAs associated with activation of the innate immune response. A panel of 159 lincRNAs was found to be differentially expressed following innate activation of THP1 macrophages. Among them, linc1992 was shown to be expressed in many human tissues and was required for induction of TNFalpha expression. Linc1992 bound specifically to heterogenous nuclear ribonucleoprotein L (hnRNPL) and formed a functional linc1992-hnRNPL complex that regulated transcription of the TNFalpha gene by binding to its promoter. Transcriptome analysis revealed that linc1992 was required for expression of many immune-response genes, including other cytokines and transcriptional and posttranscriptional regulators of TNFalpha expression, and that knockdown of linc1992 caused dysregulation of these genes during innate activation of THP1 macrophages. Therefore, we named linc1992 THRIL (TNFalpha and hnRNPL related immunoregulatory LincRNA). Finally, THRIL expression was correlated with the severity of symptoms in patients with Kawasaki disease, an acute inflammatory disease of childhood. Collectively, our data provide evidence that lincRNAs and their binding proteins can regulate TNFalpha expression and may play important roles in the innate immune response and inflammatory diseases in humans

Targeting Glycosylation Pathways and the Cell Cycle: Sugar-Dependent Activity of Butyrate-Carbohydrate Cancer Prodrugs

SummaryShort-chain fatty acid (SCFA)-carbohydrate hybrid molecules that target both histone deacetylation and glycosylation pathways to achieve sugar-dependent activity against cancer cells are described in this article. Specifically, n-butyrate esters of N-acetyl-d-mannosamine (But4ManNAc, 1) induced apoptosis, whereas corresponding N-acetyl-d-glucosamine (But4GlcNAc, 2), d-mannose (But5Man, 3), or glycerol (tributryin, 4) derivatives only provided transient cell cycle arrest. Western blots, reporter gene assays, and cell cycle analysis established that n-butyrate, when delivered to cells via any carbohydrate scaffold, functioned as a histone deacetylase inhibitor (HDACi), upregulated p21WAF1/Cip1 expression, and inhibited proliferation. However, only 1, a compound that primed sialic acid biosynthesis and modulated the expression of a different set of genes compared to 3, ultimately killed the cells. These results demonstrate that the biological activity of butyrate can be tuned by sugars to improve its anticancer properties

Ultrathin Magnesium-based Coating as an Efficient Oxygen Barrier for Superconducting Circuit Materials

Scaling up superconducting quantum circuits based on transmon qubits necessitates substantial enhancements in qubit coherence time. Among the materials considered for transmon qubits, tantalum (Ta) has emerged as a promising candidate, surpassing conventional counterparts in terms of coherence time. However, the presence of an amorphous surface Ta oxide layer introduces dielectric loss, ultimately placing a limit on the coherence time. In this study, we present a novel approach for suppressing the formation of tantalum oxide using an ultrathin magnesium (Mg) capping layer deposited on top of tantalum. Synchrotron-based X-ray photoelectron spectroscopy (XPS) studies demonstrate that oxide is confined to an extremely thin region directly beneath the Mg/Ta interface. Additionally, we demonstrate that the superconducting properties of thin Ta films are improved following the Mg capping, exhibiting sharper and higher-temperature transitions to superconductive and magnetically ordered states. Based on the experimental data and computational modeling, we establish an atomic-scale mechanistic understanding of the role of the capping layer in protecting Ta from oxidation. This work provides valuable insights into the formation mechanism and functionality of surface tantalum oxide, as well as a new materials design principle with the potential to reduce dielectric loss in superconducting quantum materials. Ultimately, our findings pave the way for the realization of large-scale, high-performance quantum computing systems

Differential sensitivity of target genes to translational repression by miR-17~92

MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5’UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genesCX is a Pew Scholar in Biomedical Sciences. This study is supported by the PEW Charitable Trusts, Cancer Research Institute, National Institute of Health (R01AI087634, R01AI089854, RC1CA146299, R56AI110403, and R01AI121155 to CX), National Natural Science Foundation of China (31570882 to WHL, 31570883 to NX, 31570911 to GF, 91429301 to JH, 31671428 and 31500665 to YZ), 1000 Young Talents Program of China (K08008 to NX), 100 Talents Program of The Chinese Academy of Sciences (YZ), National Program on Key Basic Research Project of China (2016YFA0501900 to YZ), the Fundamental Research Funds for the Central Universities of China (20720150065 to NX and GF), Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2015R1C1A1A01052387 to SGK, NRF-2016R1A4A1010115 to SGK and PHK), and 2016 Research Grant from Kangwon National University (SGK)