Search

Ribosomal RNAs (rRNAs) are extremely abundant, often constituting 80% to 90% of total RNA. Since rRNA sequences are often not of interest in genomic RNA sequencing experiments, rRNAs can be removed from the sample before the library preparation step, in order to prevent the majority of the library and the majority of sequencing reads from being rRNA. Removal of rRNA can be especially challenging for low quality and formalin-fixed paraffin-embedded (FFPE) RNA samples due to the fragmented nature of these RNA molecules. The NEBNext rRNA Depletion Kit (Human/Mouse/Rat) depletes both cytoplasmic (5 S rRNA, 5.8 S rRNA, 18 S rRNA, and 28 S rRNA) and mitochondrial rRNA (12 S rRNA and 16 S rRNA) from total RNA preparations from human, mouse, and rat samples. Due to the high similarity among mammalian rRNA sequences, it is likely that rRNA depletion can also be achieved for other mammals but has not been empirically tested. This product is compatible with both intact and degraded RNA (e.g., FFPE RNA). The resulting rRNA-depleted RNA is suitable for RNA-seq, random-primed cDNA synthesis, or other downstream RNA analysis applications. Regardless of the quality or amount of input RNA, this method efficiently removes rRNA, while retaining non-coding and other non-poly(A) RNAs. The NEBNext rRNA Depletion Kit thus provides a more complete picture of the transcript repertoire than oligo d(T) poly(A) mRNA enrichment methods. © 2016 by John Wiley & Sons, Inc. Keywords: NGS; RNA depletion; sequencing; formalin-fixed paraffin-embedded; FFPE; RNA; rRNA; ribosomal RNA; library preparation; transcriptome; rRNA removal

Neutrophils rely on exocytosis to mobilize receptors and adhesion molecules and to release microbicidal factors. This process should be strictly regulated because uncontrolled release of toxic proteins would be injurious to the host. In vivo studies showed that the small GTPase Rab27a regulates azurophilic granule exocytosis. Using mouse neutrophils deficient in Rab27a (Rab27a(ash/ash)), Rab27b [Rab27b knockout (KO)] or both [Rab27a/b double KO (DoKo)], we investigated the role of the Rab27 isoforms in neutrophils. We found that both Rab27a and Rab27b deficiencies impaired azurophilic granule exocytosis. Rab27a(ash/ash) neutrophils showed upregulation of Rab27b expression which did not compensate for the secretory defects observed in Rab27a-deficient cells, suggesting that Rab27 isoforms play independent roles in neutrophil exocytosis. Total internal reflection fluorescence microscopy analysis showed that Rab27a(ash/ash) and Rab27b KO neutrophils have a decreased number of azurophilic granules near the plasma membrane. The effect was exacerbated in Rab27a/b DoKo neutrophils. Rab27-deficient neutrophils showed impaired activation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase at the plasma membrane although intraphagosomal reactive oxygen species (ROS) production was not affected. Exocytosis of secretory vesicles in Rab27-deficient neutrophils was functional, suggesting that Rab27 GTPases selectively control the exocytosis of neutrophil granules.

Neutrophils kill bacteria on extracellular complexes of DNA fibers and bactericidal proteins known as neutrophil extracellular traps (NETs). The NET composition and the bactericidal mechanisms they use are not fully understood. Here, we show that treatment with deoxyribonuclease (DNase I) impairs a late oxidative response elicited by Gram-positive and Gram-negative bacteria and also by phorbol ester. Isoluminol-dependent chemiluminescence elicited by opsonized Listeria monocytogenes-stimulated neutrophils was inhibited by DNase I, and the DNase inhibitory effect was also evident when phagocytosis was blocked, suggesting that DNase inhibits an extracellular mechanism of reactive oxygen species (ROS) generation. The DNase inhibitory effect was independent of actin polymerization. Phagocytosis and cell viability were not impaired by DNase I. Immunofluorescence analysis shows that myeloperoxidase is present on NETs. Furthermore, granular proteins were detected in NETs from Rab27a-deficient neutrophils which have deficient exocytosis, suggesting that exocytosis and granular protein distribution on NETs proceed by independent mechanisms. NADPH oxidase subunits were also detected on NETs, and the detection of extracellular trap-associated NADPH oxidase subunits was abolished by treatment with DNase I and dependent on cell stimulation. In vitro analyses demonstrate that MPO and NADPH oxidase activity are not directly inhibited by DNase I, suggesting that its effect on ROS production depends on NET disassembly. Altogether, our data suggest that inhibition of ROS production by microorganism-derived DNase would contribute to their ability to evade killing.

Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.

Neutrophils kill micro-organisms using microbicidal products that they release into the phagosome or into the extracellular space. The secretory machinery utilized by neutrophils is poorly characterized. We show that the small GTPase Rab27a is an essential component of the secretory machinery of azurophilic granules in granulocytes. Rab27a-deficient mice have impaired secretion of MPO (myeloperoxidase) into the plasma in response to lipopolysaccharide. Cell fractionation analysis revealed that Rab27a and the Rab27a effector protein JFC1/Slp1 (synaptotagmin-like protein 1) are distributed principally in the low-density fraction containing a minor population of MPO-containing granules. By immunofluorescence microscopy, we detected Rab27a and JFC1/Slp1 in a minor subpopulation of MPO-containing granules. Interference with the JFC1/Slp1–Rab27a secretory machinery impaired secretion of MPO in permeabilized neutrophils. The expression of Rab27a was dramatically increased when promyelocytic HL-60 cells were differentiated into granulocytes but not when they were differentiated into monocytes. Down-regulation of Rab27a in HL-60 cells by RNA interference did not affect JFC1/Slp1 expression but significantly decreased the secretion of MPO. Neither Rab27a nor JFC1/Slp1 was integrated into the phagolysosome membrane during phagocytosis. Neutrophils from Rab27a-deficient mice efficiently phagocytose zymosan opsonized particles and deliver MPO to the phagosome. We conclude that Rab27a and JFC1/Slp1 permit MPO release into the surrounding milieu and constitute key components of the secretory machinery of azurophilic granules in granulocytes. Our results suggest that the granules implicated in cargo release towards the surrounding milieu are molecularly and mechanistically different from those involved in their release towards the phagolysosome.

RNA-seq has become the most popular method for transcriptome analysis and is widely used to study gene expression, detect mutations, fusion transcripts, alternative splicing, and post-transcriptional modifications. It is becoming the method of choice to detect alterations in diseases, especially cancer, to provide insights on the various molecular pathways perturbed by changes in the transcriptome and study their implications. As RNA-seq is being adopted in molecular diagnostics and biomarker identifications, the need for good quality, reproducible library preparation methods using very low amounts of RNA input, or precious clinical samples, is increasing. To address these challenges, we have developed a strand-specific RNA-seq library preparation method that retains information about which strand of DNA is transcribed, from as low as 5 ng total RNA input. Strand specificity is important for correct annotation of genes, identification of antisense transcripts with potential regulatory roles, and accurate determination of gene expression levels in the presence of antisense transcripts. Enhanced sensitivity to detect transcripts with even coverage across their length offers a non-biased approach for accurate quantification of transcript levels. Methods: Enriched poly-A mRNA or ribo-depleted RNA (Universal Human Reference RNA) was used to make libraries with our strand specific library preparation method. Library quality and quantity were analyzed on an Agilent Bioanalyzer, pooled at equimolar ratio and sequenced on Illumina’s Nextseq 500. Paired end reads were mapped to a human reference genome (hg19) using Hisat2 and sequencing metrics were calculated using Picard's RNA-seq Metrics and RSeqC tools. Transcript abundance was measured using Salmon and the Ensembl GRCh38 CDS sequences. Results: Libraries prepared with our streamlined method using inputs that range from 5ng to 1ug show greater than 98% directionality at all input levels. GC content analysis, gene body coverage and gene expression correlation are similar for all inputs tested (5ng to 1ug), even though input amounts vary by over three orders of magnitude. These consistent results are recapitulated with the spiked-in ERCC controls at all inputs. Conclusions: Our library preparation method is streamlined and can be used for a wide range of input RNA without any major modifications to the protocol, making it an easy to follow, convenient method for RNA-seq library preparation. In addition, our method has increased sensitivity and specificity, especially for low-abundance transcripts, reduced PCR duplicates and sequence bias, delivering high quality strand-specific data even for low input RNA. Finally, our method is compatible with both poly A-tail enriched and ribosomal RNA depleted samples, and is amenable to large-scale library construction and automation. Citation Format: Keerthana Krishnan, Erbay Yigit, Mehmet Karaca, Deyra Rodriguez, Bradley Langhorst, Timur Shtatland, Daniela Munafo, Pingfang Liu, Lynne Apone, Vaishnavi Panchapakesa, Karen Duggan, Christine Sumner, Christine Rozzi, Fiona Stewart, Laurie Mazzola, Joanna Bybee, Danielle Rivizzigno, Eileen T. Dimalanta, Theodore B. Davis. Low-input transcript profiling with enhanced sensitivity using a highly efficient, low-bias and strand-specific RNA-Seq library preparation method [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5406. doi:10.1158/1538-7445.AM2017-5406

Next Generation Sequencing (NGS) has significantly impacted human genetics, enabling a comprehensive characterization of the human genome as well as a better understanding of many genomic abnormalities. By delivering massive DNA sequences at unprecedented speed and cost, NGS promises to make personalized medicine a reality in the foreseeable future. To date, library construction with clinical samples has been a challenge, primarily due to the limited quantities of sample DNA available. Our objective here was to overcome this challenge by developing NEBNext® Ultra DNA Library Prep Kit, a fast library preparation method. Specifically, we streamlined the workflow utilizing novel NEBNext reagents and adaptors, including a new DNA polymerase that has been optimized to minimize GC bias. As a result of this work, we have developed a simple method for library construction from an amount of DNA as low as 5 ng, which can be used for both intact and fragmented DNA. Moreover, the workflow is compatible with multiple NGS platforms.

Background Standard RNA sequencing approaches generally require double-stranded cDNA synthesis, which erases RNA strand information. Synthesis of a randomly primed double-stranded cDNA followed by addition of adaptors for next-generation sequencing leads to the loss of information about which strand was present in the original mRNA template. The polarity of the transcript is important for correct annotation of novel genes, identification of antisense transcripts with potential regulatory roles, and for correct determination of gene expression levels in the presence of antisense transcripts. Different strand-specific RNA-seq approaches have been developed to preserve information about strand polarity with different level of performances. Material and methods Using Illumina Deep Sequencing Technology, this work investigates the performance of two different directional RNA-Seq (strand-specific RNA-seq) strategies. One is based on direct ligation of adaptors to the RNA ends and the other is based on the labeling and excision of the second strand cDNA. The RNA-seq workflows present in this work have been improved over current more laborious RNA-seq methods. Their low RNA input and streamlined workflows make them compatible with high throughput and automation. We also analyze the effect of different RNA fragmentation methods (divalent cations plus heat versus enzymatic fragmentation). Results We will provide a comparative full data analysis of different strand-specific RNA methods (library performance, complexity, continuity of gene coverage, strand specificity, rRNA background). Conclusions Our results show improved methods for high-quality strand-specific RNA-seq library construction amenable to large-scale library construction and automation.

Small regulatory RNA repertoires in biological samples are heterogeneous mixtures that may include species arising from varied biosynthetic pathways and modification events. Small RNA profiling and discovery approaches ought to capture molecules in a way that is representative of expression level. It follows that the effects of RNA modifications on representation should be minimized. The collection of high-quality, representative data, therefore, will be highly dependent on bias-free sample manipulation in advance of quantification. We examined the impact of 2′-O-methylation of the 3′-terminal nucleotide of small RNA on key enzymatic reactions of standard front-end manipulation schemes. Here we report that this common modification negatively influences the representation of these small RNA species. Deficits occurred at multiple steps as determined by gel analysis of synthetic input RNA and by quantification and sequencing of derived cDNA pools. We describe methods to minimize the effects of 2′-O-methyl modification of small RNA 3′-termini using T4 RNA ligase 2 truncated, and other optimized reaction conditions, demonstrating their use by quantifying representation of miRNAs and piRNAs in cDNA pools prepared from biological samples.

Lipopolysaccharide (LPS) stimulates exocytosis in neutrophils. The signalling molecules involved in the regulation of this mechanism are currently unknown. Using neutrophils from interleukin-1-receptor-associated kinase (IRAK)-4- and Toll/IL-1 receptor (TIR) domain-containing adaptor inducing IFN-beta (TRIF)-deficient mice, we dissected the signalling pathways that control exocytosis. We analysed exocytosis of peroxidase-negative and azurophilic granules by following the mobilization of the beta2-integrin subunit CD11b and myeloperoxidase (MPO)-containing granules, respectively. IRAK-4-null neutrophils showed marked defects in both peroxidase-negative and azurophilic granule exocytosis in response to LPS. In contrast, the exocytic response to LPS of TRIF-deficient neutrophils was not different from that of wild-type cells. No differences were observed in the exocytosis of secretory organelles between IRAK-4-null and wild-type neutrophils when they were stimulated with the phorbol ester phorbol 12-myristate 13-acetate (PMA). Electron microscopy analysis showed that no morphological abnormalities were present in the granules of IRAK-4-deficient neutrophils, suggesting that the lack of exocytic response to LPS is not attributable to developmental abnormalities. Using pharmacological inhibitors, we found that p38 mitogen-activated protein kinase (p38MAPK) is essential for the exocytosis of all neutrophil secretory organelles in response to LPS. Interestingly, we found that phosphatidylinositol 3-kinase (PI3K) is essential for azurophilic granule exocytosis but not for the mobilization of other neutrophil granules in response to LPS. Azurophilic granule exocytosis in response to Listeria monocytogenes was dependent on PI3K but not IRAK-4 activity, suggesting that alternative signalling pathways are activated in IRAK-4-deficient neutrophils exposed to whole bacteria. Our results identified IRAK-4, p38MAPK and PI3K as important regulatory components with different roles in the signalling pathways that control Toll-like receptor ligand-triggered neutrophil exocytosis.

Neutrophil granules contain secretory molecules that contribute to the implementation of all neutrophil functions. The molecular components that regulate the exocytosis of neutrophil granules have not been characterized. In this study, using small interfering RNA gene-targeting approaches and granulocytes from genetically modified mice, we characterized the Rab27a effectors JFC1/Slp1 and Munc13-4 as components of the exocytic machinery of granulocytes. Using total internal reflection fluorescence microscopy analysis, we show that Rab27a and JFC1 colocalize in predocked and docked vesicles in granulocytes. Next, we demonstrate that JFC1-downregulated granulocytes have impaired myeloperoxidase secretion. Using immunological interference, we confirm that JFC1 plays an important role in azurophilic granule exocytosis in human neutrophils. Interference with Rab27a but not with JFC1 impaired gelatinase B secretion in neutrophils, suggesting that a different Rab27a effector modulates this process. In similar studies, we confirmed that Munc13-4 regulates gelatinase secretion. Immunofluorescence analysis indicates that Munc13-4 localizes at secretory organelles in neutrophils. Using neutrophils from a Munc13-4-deficient mouse model (Jinx), we demonstrate that Munc13-4 plays a central role in the regulation of exocytosis of various sets of secretory organelles. However, mobilization of CD11b was not affected in Munc13-4-deficient neutrophils, indicating that secretory defects in these cells are limited to a selective group of exocytosable organelles.

Next generation sequencing (NGS) has significantly impacted cancer genetics, enabling a comprehensive characterization of genomic abnormalities in the cancer genome at very high resolution. By delivering massive DNA sequences at unprecedented speed and cost, NGS promises to make personalized genome-based diagnosis and targeted cancer therapy a reality in the foreseeable future. To date, library construction with clinical samples has been a challenge, primarily due to the limited quantities of sample DNA available. To overcome this challenge, we have developed a fast library preparation method using novel NEBNext reagents and adaptors. This method enables library construction from a minimal quantity of DNA (< 5 ng), and can be used for both intact and fragmented DNA. Moreover, the workflow is compatible with multiple NGS platforms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3180. doi:1538-7445.AM2012-3180

Initial transcriptomic studies largely relied on hybridization-based microarray technologies and offered a limited ability to fully catalogue and quantify the diverse RNA molecules that are expressed from genomes over wide ranges of levels. Massively parallel cDNA sequencing, or Total RNA-Seq, has allowed many advances in the characterization and quantification of transcriptomes; an offer a new appreciation for the complexity of the Transcriptome, encompassing a multitude of previously unknown coding and non-coding RNA species, particularly small RNAs, including micro RNAs. This work investigates the performance of different strategies for Total RNA-Seq using multiple next generation sequencing platforms. Standard RNA-sequencing approaches generally require double-stranded cDNA Synthesis, which erases RNA strand information. Synthesis of a randomly primed double-stranded cDNA followed by addition of adaptors for next-generation sequencing leads to the loss of information about which strand was present in the original mRNA template. The polarity of the transcript is important for correct annotation of novel genes, identification of antisense transcripts with potential regulatory roles, and for correct determination of gene expression levels in the presence of antisense transcripts. Here, we examine the performance of strand-specific RNA libraries made by direct ligation of adaptor on to the RNA. We analyze the effect of different RNA fragmentation methods (divalent cations plus heat versus enzymatic fragmentation) and we provide a comparative data analysis (library complexity, continuity of gene coverage, strand specificity and 3′and 5′-end bias analysis). Identification and analysis of small RNA by deep sequencing requires preparation of a di-tagged cDNA library, which leads to adaptor-dimer formation that strongly contaminates the library. We have developed a novel method to generate di-tagged small RNA libraries free of adapter-dimer contamination without introducing any additional enzymatic steps or gel purifications. This method has optimized the 3′adaptor-ligation reaction to recover and to increase representation of the 2′-O-modified RNAs present in a biological sample. To reduce cost and increase sample throughput we have developed a barcode strategy to tag samples during library construction. The multiplexed libraries can then be pooled together before size selection, reducing the number of steps in the workflow. This technique reduces bias by ligation, increases representation of modified small RNAs and simplifies workflow during library construction for small RNA analysis and discovery. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3186. doi:1538-7445.AM2012-3186

Background T4 RNA ligases 1 and 2 are useful tools for RNA analysis. Their use upstream of RNA analyses such as high-throughput RNA sequencing and microarrays has recently increased their importance. The truncated form of T4 RNA ligase 2, comprising amino acids 1-249 (T4 Rnl2tr), is an attractive tool for attachment of adapters or labels to RNA 3'-ends. Compared to T4 RNA ligase 1, T4 Rnl2tr has a decreased ability to ligate 5'-PO4 ends in single-stranded RNA ligations, and compared to the full-length T4 Rnl2, the T4 Rnl2tr has an increased activity for joining 5'-adenylated adapters to RNA 3'-ends. The combination of these properties allows adapter attachment to RNA 3'-ends with reduced circularization and concatemerization of substrate RNA. Results With the aim of further reducing unwanted side ligation products, we substituted active site residues, known to be important for adenylyltransferase steps of the ligation reaction, in the context of T4 Rnl2tr. We characterized the variant ligases for the formation of unwanted ligation side products and for activity in the strand-joining reaction. Conclusions Our data demonstrate that lysine 227 is a key residue facilitating adenylyl transfer from adenylated ligation donor substrates to the ligase. This reversal of the second step of the ligation reaction correlates with the formation of unwanted ligation products. Thus, T4 Rn2tr mutants containing the K227Q mutation are useful for reducing undesired ligation products. We furthermore report optimal conditions for the use of these improved T4 Rnl2tr variants.

Background Neutrophils are non-dividing cells with poor survival after isolation. Consequently, exogenous gene expression in neutrophils is challenging. We report here the transfection of genes and expression of active proteins in human primary peripheral neutrophils using nucleofection. Results Exogenous gene expression in human neutrophils was achieved 2 h post-transfection. We show that neutrophils transfected by nucleofection are functional cells, able to respond to soluble and particulate stimuli. They conserved the ability to undergo physiological processes including phagocytosis. Using this technique, we were able to show that the phox homology (PX) domain of p47 phox localizes to the plasma membrane in human neutrophils. We also show that RhoB, but not the PX domain of p47 phox , is translocated to the membrane of mature phagosomes. Conclusion We demonstrated that cDNA transfer and expression of exogenous protein in human neutrophils is compatible with cell viability and is no longer a limitation for the study of protein function in human neutrophils.

Neutrophils kill microorganisms using microbicidal products that they release into the phagosome or the extracellular space. Mature neutrophils contain four types of exocytosable storage organelles: azurophilic granules that contain myeloperoxidase, specific granules, gelatinase granules and secretory vesicles. Since the uncontrolled release of the contents of these organelles is potentially harmful, granule exocytosis must be tightly regulated. However, the secretory machinery utilized by neutrophils is poorly characterized. Here, we evaluate the role of Rab27a and its effector JFC1 in the regulated secretion of granulocytes. First, we show that JFC1 and Rab27a are highly expressed in human neutrophils. Furthermore, in HL-60 promyelocytic cells, the expression of JFC1 and Rab27a dramatically increased when they are differentiated to neutrophil-like granulocytes by DMSO treatment, supporting a role for these proteins in the secretory machinery of granulocytes. We found that while Rab27a distribution is restricted to the membrane fraction after cell fractionation, JFC1 is distributed between the membrane fraction and the cytosol. The localization of Rab27a in neutrophils was confirmed by immuno-electron microscopy. Colocalization of endogenous JFC1 and Rab27a was observed by immunofluorescence analysis. After cell fractionation, JFC1 and Rab27a were mainly associated with the tertiary granules (γ fraction) which are enriched in MMP-9 and cytochrome b558. A small proportion of the myeloperoxidase-positive granules were also detected in this fraction. Both Rab27a and JFC1 colocalized with VAMP2, a marker of secretory vesicles. The introduction of the plasma membrane binding domain (C2A domain) of JFC1 into permeabilized neutrophils significantly decreased exocytosis, however, neither Rab27a nor JFC1 integrated to the phagolysosome when neutrophils were exposed to opsonized particles, suggesting that the granules implicated in cargo release towards the surrounding milieu are molecularly and mechanistically different from those involved in their release towards the phagolysosome.