ultra directional rna library prep kit  (New England Biolabs)


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    New England Biolabs ultra directional rna library prep kit
    Ultra Directional Rna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 558 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ultra directional rna library prep kit/product/New England Biolabs
    Average 99 stars, based on 558 article reviews
    Price from $9.99 to $1999.99
    ultra directional rna library prep kit - by Bioz Stars, 2020-04
    99/100 stars

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    Related Articles

    RNA Extraction:

    Article Title: Identification and complete genome analysis of a novel bovine picornavirus in Japan.
    Article Snippet: .. Viral RNA extraction, cDNA library construction, and deep sequencing Viral RNAs were extracted from the filtrates using TRIzol ® LS Reagent (Life technologies, Carlsbad, CA, USA) and treated with DNase I (Takara Bio, Shiga, Japan). cDNA libraries for deep sequencing were constructed from RNA using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's guidelines. .. Whole genome sequencing was determined by deep sequencing and the rapid amplification cDNA end method (RACE) (Roche Diagnostics GmbH, Mannheim, Germany) and 5 -Full RACE Core Set (Takara Bio).

    Article Title: The regulation mechanism of lncRNAs and mRNAs in sea cucumbers under global climate changes: Defense against thermal and hypoxic stresses.
    Article Snippet: Library preparation and examination for sequencing Total RNA from tissues was extracted using MiniBEST Universal RNA Extraction Kit (Takara, Shiga, Japan) following the manufacturer's recommendations. .. The sequencing libraries were constructed with varied index labels by a NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, USA) , and the details are as follows: first, ribosomal RNA was removed utilizing Epicentre Ribo-Zero™ Gold Kits (Epicentre, USA), and then NEBNext First-Strand Synthesis Reaction Buffer under raised temperature was used to fracture RNA into short RNA strands.

    Amplification:

    Article Title: Identification and complete genome analysis of a novel bovine picornavirus in Japan.
    Article Snippet: Viral RNA extraction, cDNA library construction, and deep sequencing Viral RNAs were extracted from the filtrates using TRIzol ® LS Reagent (Life technologies, Carlsbad, CA, USA) and treated with DNase I (Takara Bio, Shiga, Japan). cDNA libraries for deep sequencing were constructed from RNA using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's guidelines. .. Whole genome sequencing was determined by deep sequencing and the rapid amplification cDNA end method (RACE) (Roche Diagnostics GmbH, Mannheim, Germany) and 5 -Full RACE Core Set (Takara Bio).

    Article Title: The transcriptional repressor SNAI2 impairs neuroblastoma differentiation and inhibits response to retinoic acid therapy.
    Article Snippet: Samples were processed using the NEBNext® Ultra™ Directional RNA Library Prep kit for Illumina® (#E7420S, NEB) according to manufactures instructions. .. In short, after isolation of mRNA from total RNA using the oligo-dT magnetic beads, mRNA was fragmented and cDNA synthesis was performed for ligation with the sequencing adapters and PCR amplification of the resulting product.

    Sample Prep:

    Article Title: The transcriptional repressor SNAI2 impairs neuroblastoma differentiation and inhibits response to retinoic acid therapy.
    Article Snippet: Paragraph title: 2.7.1. Sample preparation ... Samples were processed using the NEBNext® Ultra™ Directional RNA Library Prep kit for Illumina® (#E7420S, NEB) according to manufactures instructions.

    Magnetic Beads:

    Article Title: The transcriptional repressor SNAI2 impairs neuroblastoma differentiation and inhibits response to retinoic acid therapy.
    Article Snippet: Samples were processed using the NEBNext® Ultra™ Directional RNA Library Prep kit for Illumina® (#E7420S, NEB) according to manufactures instructions. .. In short, after isolation of mRNA from total RNA using the oligo-dT magnetic beads, mRNA was fragmented and cDNA synthesis was performed for ligation with the sequencing adapters and PCR amplification of the resulting product.

    Isolation:

    Article Title: Retrospective Validation of a Metagenomic Sequencing Protocol for Combined Detection of RNA and DNA Viruses Using Respiratory Samples from Pediatric Patients.
    Article Snippet: Library Preparation Libraries were constructed with 7 mL extracted nucleic acids using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA) using single, unique adaptors. .. The following steps were omitted: poly A mRNA capture isolation (instruction manual New England Biolabs number E7420S/L, version 8.0, chapter 1), rRNA depletion, and DNase step (chapter 2.1 to 2.4, 2.5B, 2.11A).

    Article Title: The transcriptional repressor SNAI2 impairs neuroblastoma differentiation and inhibits response to retinoic acid therapy.
    Article Snippet: Samples were processed using the NEBNext® Ultra™ Directional RNA Library Prep kit for Illumina® (#E7420S, NEB) according to manufactures instructions. .. In short, after isolation of mRNA from total RNA using the oligo-dT magnetic beads, mRNA was fragmented and cDNA synthesis was performed for ligation with the sequencing adapters and PCR amplification of the resulting product.

    Ligation:

    Article Title: The regulation mechanism of lncRNAs and mRNAs in sea cucumbers under global climate changes: Defense against thermal and hypoxic stresses.
    Article Snippet: The sequencing libraries were constructed with varied index labels by a NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, USA) , and the details are as follows: first, ribosomal RNA was removed utilizing Epicentre Ribo-Zero™ Gold Kits (Epicentre, USA), and then NEBNext First-Strand Synthesis Reaction Buffer under raised temperature was used to fracture RNA into short RNA strands. .. Later on, terminal repair, poly(A) tailing and adapter ligation were implemented.

    Article Title: The transcriptional repressor SNAI2 impairs neuroblastoma differentiation and inhibits response to retinoic acid therapy.
    Article Snippet: Samples were processed using the NEBNext® Ultra™ Directional RNA Library Prep kit for Illumina® (#E7420S, NEB) according to manufactures instructions. .. In short, after isolation of mRNA from total RNA using the oligo-dT magnetic beads, mRNA was fragmented and cDNA synthesis was performed for ligation with the sequencing adapters and PCR amplification of the resulting product.

    Construct:

    Article Title: Retrospective Validation of a Metagenomic Sequencing Protocol for Combined Detection of RNA and DNA Viruses Using Respiratory Samples from Pediatric Patients.
    Article Snippet: .. Library Preparation Libraries were constructed with 7 mL extracted nucleic acids using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA) using single, unique adaptors. ..

    Article Title: Identification and complete genome analysis of a novel bovine picornavirus in Japan.
    Article Snippet: .. Viral RNA extraction, cDNA library construction, and deep sequencing Viral RNAs were extracted from the filtrates using TRIzol ® LS Reagent (Life technologies, Carlsbad, CA, USA) and treated with DNase I (Takara Bio, Shiga, Japan). cDNA libraries for deep sequencing were constructed from RNA using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's guidelines. .. Whole genome sequencing was determined by deep sequencing and the rapid amplification cDNA end method (RACE) (Roche Diagnostics GmbH, Mannheim, Germany) and 5 -Full RACE Core Set (Takara Bio).

    Article Title: The regulation mechanism of lncRNAs and mRNAs in sea cucumbers under global climate changes: Defense against thermal and hypoxic stresses.
    Article Snippet: .. The sequencing libraries were constructed with varied index labels by a NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, USA) , and the details are as follows: first, ribosomal RNA was removed utilizing Epicentre Ribo-Zero™ Gold Kits (Epicentre, USA), and then NEBNext First-Strand Synthesis Reaction Buffer under raised temperature was used to fracture RNA into short RNA strands. .. Subsequently, random hexamer primers were used to synthesize the firststrand cDNA by utilizing RNA fragments as a template.

    Purification:

    Article Title: The regulation mechanism of lncRNAs and mRNAs in sea cucumbers under global climate changes: Defense against thermal and hypoxic stresses.
    Article Snippet: The sequencing libraries were constructed with varied index labels by a NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, USA) , and the details are as follows: first, ribosomal RNA was removed utilizing Epicentre Ribo-Zero™ Gold Kits (Epicentre, USA), and then NEBNext First-Strand Synthesis Reaction Buffer under raised temperature was used to fracture RNA into short RNA strands. .. Afterwards, second-strand cDNA synthesis was performed using a buffer, dNTPs, DNA polymerase I and RNase H. Then the library fragments were purified with QIAQuick PCR kits, and eluted with elution buffer (EB).

    Article Title: The transcriptional repressor SNAI2 impairs neuroblastoma differentiation and inhibits response to retinoic acid therapy.
    Article Snippet: Sample preparation RNA samples of SH-SY5Y SNAI2 control and gRNA cells were prepared and analyzed as three independent experiments. mRNA was purified from cell cultures using an RNeasy minikit (#74106, Qiagen) with on column DNAse (#79254, Qiagen) treatment. .. Samples were processed using the NEBNext® Ultra™ Directional RNA Library Prep kit for Illumina® (#E7420S, NEB) according to manufactures instructions.

    Concentration Assay:

    Article Title: The regulation mechanism of lncRNAs and mRNAs in sea cucumbers under global climate changes: Defense against thermal and hypoxic stresses.
    Article Snippet: The RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA) was used for RNA integrity and concentration assessment. .. The sequencing libraries were constructed with varied index labels by a NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, USA) , and the details are as follows: first, ribosomal RNA was removed utilizing Epicentre Ribo-Zero™ Gold Kits (Epicentre, USA), and then NEBNext First-Strand Synthesis Reaction Buffer under raised temperature was used to fracture RNA into short RNA strands.

    Random Hexamer Labeling:

    Article Title: The regulation mechanism of lncRNAs and mRNAs in sea cucumbers under global climate changes: Defense against thermal and hypoxic stresses.
    Article Snippet: The sequencing libraries were constructed with varied index labels by a NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, USA) , and the details are as follows: first, ribosomal RNA was removed utilizing Epicentre Ribo-Zero™ Gold Kits (Epicentre, USA), and then NEBNext First-Strand Synthesis Reaction Buffer under raised temperature was used to fracture RNA into short RNA strands. .. Subsequently, random hexamer primers were used to synthesize the firststrand cDNA by utilizing RNA fragments as a template.

    Polymerase Chain Reaction:

    Article Title: The regulation mechanism of lncRNAs and mRNAs in sea cucumbers under global climate changes: Defense against thermal and hypoxic stresses.
    Article Snippet: The sequencing libraries were constructed with varied index labels by a NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, USA) , and the details are as follows: first, ribosomal RNA was removed utilizing Epicentre Ribo-Zero™ Gold Kits (Epicentre, USA), and then NEBNext First-Strand Synthesis Reaction Buffer under raised temperature was used to fracture RNA into short RNA strands. .. Afterwards, second-strand cDNA synthesis was performed using a buffer, dNTPs, DNA polymerase I and RNase H. Then the library fragments were purified with QIAQuick PCR kits, and eluted with elution buffer (EB).

    Article Title: The transcriptional repressor SNAI2 impairs neuroblastoma differentiation and inhibits response to retinoic acid therapy.
    Article Snippet: Samples were processed using the NEBNext® Ultra™ Directional RNA Library Prep kit for Illumina® (#E7420S, NEB) according to manufactures instructions. .. In short, after isolation of mRNA from total RNA using the oligo-dT magnetic beads, mRNA was fragmented and cDNA synthesis was performed for ligation with the sequencing adapters and PCR amplification of the resulting product.

    cDNA Library Assay:

    Article Title: Identification and complete genome analysis of a novel bovine picornavirus in Japan.
    Article Snippet: .. Viral RNA extraction, cDNA library construction, and deep sequencing Viral RNAs were extracted from the filtrates using TRIzol ® LS Reagent (Life technologies, Carlsbad, CA, USA) and treated with DNase I (Takara Bio, Shiga, Japan). cDNA libraries for deep sequencing were constructed from RNA using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's guidelines. .. Whole genome sequencing was determined by deep sequencing and the rapid amplification cDNA end method (RACE) (Roche Diagnostics GmbH, Mannheim, Germany) and 5 -Full RACE Core Set (Takara Bio).

    Spectrophotometry:

    Article Title: The regulation mechanism of lncRNAs and mRNAs in sea cucumbers under global climate changes: Defense against thermal and hypoxic stresses.
    Article Snippet: RNA degradation and purity were detected using 1% agarose gels and kaiaoK5500® spectrophotometer (Kaiao, Beijing, China), respectively. .. The sequencing libraries were constructed with varied index labels by a NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, USA) , and the details are as follows: first, ribosomal RNA was removed utilizing Epicentre Ribo-Zero™ Gold Kits (Epicentre, USA), and then NEBNext First-Strand Synthesis Reaction Buffer under raised temperature was used to fracture RNA into short RNA strands.

    Sequencing:

    Article Title: Identification and complete genome analysis of a novel bovine picornavirus in Japan.
    Article Snippet: .. Viral RNA extraction, cDNA library construction, and deep sequencing Viral RNAs were extracted from the filtrates using TRIzol ® LS Reagent (Life technologies, Carlsbad, CA, USA) and treated with DNase I (Takara Bio, Shiga, Japan). cDNA libraries for deep sequencing were constructed from RNA using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's guidelines. .. Whole genome sequencing was determined by deep sequencing and the rapid amplification cDNA end method (RACE) (Roche Diagnostics GmbH, Mannheim, Germany) and 5 -Full RACE Core Set (Takara Bio).

    Article Title: The regulation mechanism of lncRNAs and mRNAs in sea cucumbers under global climate changes: Defense against thermal and hypoxic stresses.
    Article Snippet: .. The sequencing libraries were constructed with varied index labels by a NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, USA) , and the details are as follows: first, ribosomal RNA was removed utilizing Epicentre Ribo-Zero™ Gold Kits (Epicentre, USA), and then NEBNext First-Strand Synthesis Reaction Buffer under raised temperature was used to fracture RNA into short RNA strands. .. Subsequently, random hexamer primers were used to synthesize the firststrand cDNA by utilizing RNA fragments as a template.

    Article Title: The transcriptional repressor SNAI2 impairs neuroblastoma differentiation and inhibits response to retinoic acid therapy.
    Article Snippet: Samples were processed using the NEBNext® Ultra™ Directional RNA Library Prep kit for Illumina® (#E7420S, NEB) according to manufactures instructions. .. In short, after isolation of mRNA from total RNA using the oligo-dT magnetic beads, mRNA was fragmented and cDNA synthesis was performed for ligation with the sequencing adapters and PCR amplification of the resulting product.

    DNA Sequencing:

    Article Title: The transcriptional repressor SNAI2 impairs neuroblastoma differentiation and inhibits response to retinoic acid therapy.
    Article Snippet: Samples were processed using the NEBNext® Ultra™ Directional RNA Library Prep kit for Illumina® (#E7420S, NEB) according to manufactures instructions. .. Clustering and DNA sequencing using the Illumina cBot and HiSeq 4000 was performed according to manufacturer's protocols using DNA concentrations of 3.0 nM.

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    New England Biolabs ultra directional rna library prep kit
    Internode zones have unique <t>RNAseq</t> profiles and contain stem- and zone-specific genes. a Principal component analysis of FPKM values obtained from <t>RNA</t> sequencing of the MsZ, CEZ, TZ, and MZ of internode 5. b Venn diagram of genes up-regulated greater than a log 2 fold change of one against the background levels in the whole plant (WP) transcriptome. c Table showing the number of genes expressed > 1 FPKM, number of genes up-regulated against the WP transcriptome with > 1 log 2 fold, number of identified ‘stem-specific’ genes with > 2.5 log 2 fold change against the WP background and > 80 FPKM in any internode zone, and the number of ‘zone-specific’ genes with, in addition to being ‘stem-specific’, > 2 log 2 fold change against all other internodal zones (with some exceptions, see Additional file 2 )
    Ultra Directional Rna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 558 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ultra directional rna library prep kit/product/New England Biolabs
    Average 99 stars, based on 558 article reviews
    Price from $9.99 to $1999.99
    ultra directional rna library prep kit - by Bioz Stars, 2020-04
    99/100 stars
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    Internode zones have unique RNAseq profiles and contain stem- and zone-specific genes. a Principal component analysis of FPKM values obtained from RNA sequencing of the MsZ, CEZ, TZ, and MZ of internode 5. b Venn diagram of genes up-regulated greater than a log 2 fold change of one against the background levels in the whole plant (WP) transcriptome. c Table showing the number of genes expressed > 1 FPKM, number of genes up-regulated against the WP transcriptome with > 1 log 2 fold, number of identified ‘stem-specific’ genes with > 2.5 log 2 fold change against the WP background and > 80 FPKM in any internode zone, and the number of ‘zone-specific’ genes with, in addition to being ‘stem-specific’, > 2 log 2 fold change against all other internodal zones (with some exceptions, see Additional file 2 )

    Journal: Biotechnology for Biofuels

    Article Title: A developing Setaria viridis internode: an experimental system for the study of biomass generation in a C4 model species

    doi: 10.1186/s13068-016-0457-6

    Figure Lengend Snippet: Internode zones have unique RNAseq profiles and contain stem- and zone-specific genes. a Principal component analysis of FPKM values obtained from RNA sequencing of the MsZ, CEZ, TZ, and MZ of internode 5. b Venn diagram of genes up-regulated greater than a log 2 fold change of one against the background levels in the whole plant (WP) transcriptome. c Table showing the number of genes expressed > 1 FPKM, number of genes up-regulated against the WP transcriptome with > 1 log 2 fold, number of identified ‘stem-specific’ genes with > 2.5 log 2 fold change against the WP background and > 80 FPKM in any internode zone, and the number of ‘zone-specific’ genes with, in addition to being ‘stem-specific’, > 2 log 2 fold change against all other internodal zones (with some exceptions, see Additional file 2 )

    Article Snippet: RNAseq libraries were generated with polyA enrichment and rRNA depletion using NEBNext® Ultra™ Directional RNA Library Prep Kit (NEB, Ipswich, USA, http://www.neb.com ) for Illumina® and were sequenced on an Illumina® HiSeq 2500 at the Max Planck Genome Centre, Cologne, Germany.

    Techniques: RNA Sequencing Assay

    Presence of stable miRNAs in Drosophila melanogaster hemolymph. ( A , B ) Clear HL droplets extruded from fly head and thorax. ( C – F ) Real-time qPCR amplification of selected HL miRNAs and mRNAs. Total RNA including small RNA was extracted from HL samples and analyzed with qPCR to measure the levels of miRNAs and mRNAs. The y -axis represents the relative fluorescence units (RFU) in a semi-log scale. The x -axis represents the cycle at which fluorescence was detected above an automatically determined threshold. ( C ) Amplification plots for miR-14, miR-8, and miR-184 measured in a representative HL sample. ( D ) Amplification plots for miR-14, let-7, bantam, and spiked-in synthetic C. elegans cel-miR-39 RNA in representative HL sample. ( E ) The amplification plots for tubulin, actin, and gapdh mRNAs determined by qPCR using total RNA from HL and S2 cells. Sample from S2 cells are used as a positive control for detecting Drosophila mRNAs by qPCR. The amplification curves of all three mRNAs are superimposed on one another, reflecting the presence of similar amounts of these mRNA in S2 cells. Amplification curves from HL samples show that fluorescent products appear after about 30 cycles, reflecting the significantly lower abundance of these mRNAs in HL relative to S2 cells. ( F ) The cycle threshold (Ct) fold-change of selected miRNA amplified in the absence or presence of RNase A and DNase I. Total RNA was extracted from HL samples spiked with 10 fmoles of cel-miR-39 RNA. The x -axis represents the ratio of raw Ct values from control samples divided by raw Ct values from samples incubated with RNase A and DNase I. The significantly higher magnitude of the Ct fold change of the spiked-in synthetic miRNA relative to those of the miRNA indicates that the HL-miRNA are present in nuclease-resistant, stable form.

    Journal: Genomics Insights

    Article Title: MicroRNAs Circulate in the Hemolymph of Drosophila and Accumulate Relative to Tissue microRNAs in an Age-Dependent Manner

    doi: 10.4137/GEI.S38147

    Figure Lengend Snippet: Presence of stable miRNAs in Drosophila melanogaster hemolymph. ( A , B ) Clear HL droplets extruded from fly head and thorax. ( C – F ) Real-time qPCR amplification of selected HL miRNAs and mRNAs. Total RNA including small RNA was extracted from HL samples and analyzed with qPCR to measure the levels of miRNAs and mRNAs. The y -axis represents the relative fluorescence units (RFU) in a semi-log scale. The x -axis represents the cycle at which fluorescence was detected above an automatically determined threshold. ( C ) Amplification plots for miR-14, miR-8, and miR-184 measured in a representative HL sample. ( D ) Amplification plots for miR-14, let-7, bantam, and spiked-in synthetic C. elegans cel-miR-39 RNA in representative HL sample. ( E ) The amplification plots for tubulin, actin, and gapdh mRNAs determined by qPCR using total RNA from HL and S2 cells. Sample from S2 cells are used as a positive control for detecting Drosophila mRNAs by qPCR. The amplification curves of all three mRNAs are superimposed on one another, reflecting the presence of similar amounts of these mRNA in S2 cells. Amplification curves from HL samples show that fluorescent products appear after about 30 cycles, reflecting the significantly lower abundance of these mRNAs in HL relative to S2 cells. ( F ) The cycle threshold (Ct) fold-change of selected miRNA amplified in the absence or presence of RNase A and DNase I. Total RNA was extracted from HL samples spiked with 10 fmoles of cel-miR-39 RNA. The x -axis represents the ratio of raw Ct values from control samples divided by raw Ct values from samples incubated with RNase A and DNase I. The significantly higher magnitude of the Ct fold change of the spiked-in synthetic miRNA relative to those of the miRNA indicates that the HL-miRNA are present in nuclease-resistant, stable form.

    Article Snippet: One-microgram aliquots of total RNA were used to construct mRNA sequencing libraries (mRNA-Seq) using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, #E7420S).

    Techniques: Real-time Polymerase Chain Reaction, Amplification, Fluorescence, Positive Control, Incubation

    Outline of the experimental design and workflow for the integrated analysis of the miRNA-Seq and mRNA-Seq data. Genes that are potentially regulated by the HL-miRNAs were determined by integrating miRNA-Seq and mRNA-Seq data using the following three steps: Step 1 (depicted in green): After identifying the HL-miRNAs enriched relative to BT in either young or old, or both young and old ( Tables 1 and 2 ), we retrieved their computationally predicted target genes from the m 3 RNA database ( http://m3rna.cnb.csic.es ). Step 2 (depicted in blue): mRNA-Seq was utilized to identify three groups of genes that were differentially expressed with age, either upregulated, downregulated, or unchanged in BT. Step 3 (depicted in red): The three groups of genes predicted in step1 as targets of HL-miRNAs were intersected with the three groups of genes identified in step 2. In the figure, ( A ) depicts the derivation of genes that are predicted to be targets of HL-miRNAs enriched in young which are also upregulated with age in BT. These genes (Supplementary Table 1 ) are likely upregulated in BT of old flies because their expression is no longer repressed by HL-miRNAs that are enriched only in young flies. ( B ) Depicts the derivation of genes which are the predicted to be targets of HL-miRNAs enriched in old which are also downregulated with age in BT. These genes (Supplementary Table 2 ) are likely downregulated in BT of old flies because their expression is repressed by HL-miRNAs which are enriched only in old flies. ( C ) Depicts the derivation of genes which are predicted to be targets of HL-miRNAs enriched in both young and old which also do not change expression with age in BT. These genes (Supplementary Table 3) do not change expression with age because the concentration of their regulatory miRNAs in HL does not change with age.

    Journal: Genomics Insights

    Article Title: MicroRNAs Circulate in the Hemolymph of Drosophila and Accumulate Relative to Tissue microRNAs in an Age-Dependent Manner

    doi: 10.4137/GEI.S38147

    Figure Lengend Snippet: Outline of the experimental design and workflow for the integrated analysis of the miRNA-Seq and mRNA-Seq data. Genes that are potentially regulated by the HL-miRNAs were determined by integrating miRNA-Seq and mRNA-Seq data using the following three steps: Step 1 (depicted in green): After identifying the HL-miRNAs enriched relative to BT in either young or old, or both young and old ( Tables 1 and 2 ), we retrieved their computationally predicted target genes from the m 3 RNA database ( http://m3rna.cnb.csic.es ). Step 2 (depicted in blue): mRNA-Seq was utilized to identify three groups of genes that were differentially expressed with age, either upregulated, downregulated, or unchanged in BT. Step 3 (depicted in red): The three groups of genes predicted in step1 as targets of HL-miRNAs were intersected with the three groups of genes identified in step 2. In the figure, ( A ) depicts the derivation of genes that are predicted to be targets of HL-miRNAs enriched in young which are also upregulated with age in BT. These genes (Supplementary Table 1 ) are likely upregulated in BT of old flies because their expression is no longer repressed by HL-miRNAs that are enriched only in young flies. ( B ) Depicts the derivation of genes which are the predicted to be targets of HL-miRNAs enriched in old which are also downregulated with age in BT. These genes (Supplementary Table 2 ) are likely downregulated in BT of old flies because their expression is repressed by HL-miRNAs which are enriched only in old flies. ( C ) Depicts the derivation of genes which are predicted to be targets of HL-miRNAs enriched in both young and old which also do not change expression with age in BT. These genes (Supplementary Table 3) do not change expression with age because the concentration of their regulatory miRNAs in HL does not change with age.

    Article Snippet: One-microgram aliquots of total RNA were used to construct mRNA sequencing libraries (mRNA-Seq) using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, #E7420S).

    Techniques: Expressing, Concentration Assay

    Analysis of the F. novicida greA locus. ( A ) Alignment of four Gram-negative bacterial GreA amino acid sequences using the MegAlign program of the DNAstar Lasergene package (version 10). Identical amino acid residues are shown in black. The cross-link with the RNA 3′-terminus is shown in a red box and the conserved acidic residues (D43 and E46) required for GreA activity are shown in green boxes. ( B ) Schematic illustration of the gene arrangement at the greA locus. ( C ) Cotranscription of the greA locus genes determined with RT–PCR. The pepA-–guaB (a), guaB– FTN_0662 (b), FTN_0662 –pgi (c), pgi–fimT (d), fimT–greA (e), and greA–uvrA (f) junctions were amplified using DNA, DNA-free RNA, or cDNA as the template. The images were acquired by the gel imaging system (LIUYI, Beijing, China). The experiment was repeated twice. The sizes of the molecular markers are indicated at the side in kbp.

    Journal: Scientific Reports

    Article Title: Transcription Elongation Factor GreA Plays a Key Role in Cellular Invasion and Virulence of Francisella tularensis subsp. novicida

    doi: 10.1038/s41598-018-25271-5

    Figure Lengend Snippet: Analysis of the F. novicida greA locus. ( A ) Alignment of four Gram-negative bacterial GreA amino acid sequences using the MegAlign program of the DNAstar Lasergene package (version 10). Identical amino acid residues are shown in black. The cross-link with the RNA 3′-terminus is shown in a red box and the conserved acidic residues (D43 and E46) required for GreA activity are shown in green boxes. ( B ) Schematic illustration of the gene arrangement at the greA locus. ( C ) Cotranscription of the greA locus genes determined with RT–PCR. The pepA-–guaB (a), guaB– FTN_0662 (b), FTN_0662 –pgi (c), pgi–fimT (d), fimT–greA (e), and greA–uvrA (f) junctions were amplified using DNA, DNA-free RNA, or cDNA as the template. The images were acquired by the gel imaging system (LIUYI, Beijing, China). The experiment was repeated twice. The sizes of the molecular markers are indicated at the side in kbp.

    Article Snippet: The Ribo-Zero™ Magnetic Kit (Epicentre, Madison, WI, USA) was used to remove the rRNA, and the rRNA-depleted RNA was used to generate cDNA libraries with the NEBNext Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB).

    Techniques: Activity Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Imaging

    Verification of RNA-seq data. ( A ) Detection of gene transcription in the wild-type U112 strain and the Δ greA mutant. Transcription levels of genes by RNA-seq were shown with solid bars. Relative level of each target gene (open bars) by qRT-PCR was normalized to that of the 16S rRNA gene. Data are presented as mean fold changes relative to the wild-type U112 strain ± SD of the results from triplicate samples. The experiment was repeated twice. ( B ) Detection of protein expression in the wild-type U112 strain and the Δ greA mutant. Mid-log bacteria were resuspended in PBS to OD 600 = 1.0. The suspensions were concentrated 10-fold, separated with SDS-PAGE, and detected with western blotting, using antiserum specific for each target protein. The left panel is a section of a coomassie stained gel as a loading control. The coomassie stained gel image was acquired with the digital camera (Canon, Janpan). The experiment was repeated twice. Size of each protein is indicated on the left in kDa.

    Journal: Scientific Reports

    Article Title: Transcription Elongation Factor GreA Plays a Key Role in Cellular Invasion and Virulence of Francisella tularensis subsp. novicida

    doi: 10.1038/s41598-018-25271-5

    Figure Lengend Snippet: Verification of RNA-seq data. ( A ) Detection of gene transcription in the wild-type U112 strain and the Δ greA mutant. Transcription levels of genes by RNA-seq were shown with solid bars. Relative level of each target gene (open bars) by qRT-PCR was normalized to that of the 16S rRNA gene. Data are presented as mean fold changes relative to the wild-type U112 strain ± SD of the results from triplicate samples. The experiment was repeated twice. ( B ) Detection of protein expression in the wild-type U112 strain and the Δ greA mutant. Mid-log bacteria were resuspended in PBS to OD 600 = 1.0. The suspensions were concentrated 10-fold, separated with SDS-PAGE, and detected with western blotting, using antiserum specific for each target protein. The left panel is a section of a coomassie stained gel as a loading control. The coomassie stained gel image was acquired with the digital camera (Canon, Janpan). The experiment was repeated twice. Size of each protein is indicated on the left in kDa.

    Article Snippet: The Ribo-Zero™ Magnetic Kit (Epicentre, Madison, WI, USA) was used to remove the rRNA, and the rRNA-depleted RNA was used to generate cDNA libraries with the NEBNext Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB).

    Techniques: RNA Sequencing Assay, Mutagenesis, Quantitative RT-PCR, Expressing, SDS Page, Western Blot, Staining