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    New England Biolabs ultra directional rna library prep kit
    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 <t>RNA</t> 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 <t>cDNA</t> 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.
    Ultra Directional Rna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 6796 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    RNA-seq analysis of BioTAP-XL pull-downs. ( A ) Enrichment of repeat-derived RNA in HP1a-BioTAP cross-linked complexes from S2 cells compared with MSL3-BioTAP complexes from S2 cells detected using a random-priming approach for cDNA synthesis and Illumina

    Journal: Genes & Development

    Article Title: Heterochromatin-associated interactions of Drosophila HP1a with dADD1, HIPP1, and repetitive RNAs

    doi: 10.1101/gad.241950.114

    Figure Lengend Snippet: RNA-seq analysis of BioTAP-XL pull-downs. ( A ) Enrichment of repeat-derived RNA in HP1a-BioTAP cross-linked complexes from S2 cells compared with MSL3-BioTAP complexes from S2 cells detected using a random-priming approach for cDNA synthesis and Illumina

    Article Snippet: An NEBNext ultradirectional RNA library kit (New England Biolabs, catalog no. E7420S) was used to make cDNA and RNA-seq libraries.

    Techniques: RNA Sequencing Assay, Derivative Assay

    Hierarchical clustering of expression levels, based on the rank of the count of exon per million mapped reads (CPM). Dendrogram represents Spearman correlation coefficients between pairs of samples. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSe q using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples.. Color scale: Spearman correlation coefficients

    Journal: BMC Genomics

    Article Title: A comparative analysis of library prep approaches for sequencing low input translatome samples

    doi: 10.1186/s12864-018-5066-2

    Figure Lengend Snippet: Hierarchical clustering of expression levels, based on the rank of the count of exon per million mapped reads (CPM). Dendrogram represents Spearman correlation coefficients between pairs of samples. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSe q using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples.. Color scale: Spearman correlation coefficients

    Article Snippet: We compared 5 methods of library preparation for Illumina Next Generation sequencing: NuGEN Ovation RNA-Seq system V2 Kit, TaKaRa SMARTer Stranded Total RNA-Seq Kit, TaKaRa SMART-Seq v4 Ultra Low Input RNA Kit, Illumina TruSeq RNA Library Prep Kit v2 and NEBNext® Ultra™ Directional RNA Library Prep Kit using slightly modified protocols each with 4 ng of total RNA.

    Techniques: Expressing

    Hierarchical clustering based on the rank of IP/input value. Dendrogram represents Spearman correlation coefficients between pairs of samples. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples

    Journal: BMC Genomics

    Article Title: A comparative analysis of library prep approaches for sequencing low input translatome samples

    doi: 10.1186/s12864-018-5066-2

    Figure Lengend Snippet: Hierarchical clustering based on the rank of IP/input value. Dendrogram represents Spearman correlation coefficients between pairs of samples. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples

    Article Snippet: We compared 5 methods of library preparation for Illumina Next Generation sequencing: NuGEN Ovation RNA-Seq system V2 Kit, TaKaRa SMARTer Stranded Total RNA-Seq Kit, TaKaRa SMART-Seq v4 Ultra Low Input RNA Kit, Illumina TruSeq RNA Library Prep Kit v2 and NEBNext® Ultra™ Directional RNA Library Prep Kit using slightly modified protocols each with 4 ng of total RNA.

    Techniques:

    Descriptive characteristic of enrichment or depletion profiles as generated by the different library preparation kits. Genes which have at least 20 raw reads in the input samples and a ratio of IP/Input ≥2 or Input/IP ≥2 were used to generate the plots. a Total number of transcripts enriched or depleted. b Percentage of enriched or depleted transcripts grouped into different bins. X-axis: log2(IP/input), Y-axis: percentage of genes in each bin over whole population. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples

    Journal: BMC Genomics

    Article Title: A comparative analysis of library prep approaches for sequencing low input translatome samples

    doi: 10.1186/s12864-018-5066-2

    Figure Lengend Snippet: Descriptive characteristic of enrichment or depletion profiles as generated by the different library preparation kits. Genes which have at least 20 raw reads in the input samples and a ratio of IP/Input ≥2 or Input/IP ≥2 were used to generate the plots. a Total number of transcripts enriched or depleted. b Percentage of enriched or depleted transcripts grouped into different bins. X-axis: log2(IP/input), Y-axis: percentage of genes in each bin over whole population. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples

    Article Snippet: We compared 5 methods of library preparation for Illumina Next Generation sequencing: NuGEN Ovation RNA-Seq system V2 Kit, TaKaRa SMARTer Stranded Total RNA-Seq Kit, TaKaRa SMART-Seq v4 Ultra Low Input RNA Kit, Illumina TruSeq RNA Library Prep Kit v2 and NEBNext® Ultra™ Directional RNA Library Prep Kit using slightly modified protocols each with 4 ng of total RNA.

    Techniques: Generated

    Enrichment profiles and top 50 enriched transcripts. a Enrichment factor of transcripts are sorted in decreasing order based on log2 (IP/input). X-axis:transcripts, Y-axis:log2 value of enrichment (IP/Input). b Boxplot of top 50 enriched transcripts. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples

    Journal: BMC Genomics

    Article Title: A comparative analysis of library prep approaches for sequencing low input translatome samples

    doi: 10.1186/s12864-018-5066-2

    Figure Lengend Snippet: Enrichment profiles and top 50 enriched transcripts. a Enrichment factor of transcripts are sorted in decreasing order based on log2 (IP/input). X-axis:transcripts, Y-axis:log2 value of enrichment (IP/Input). b Boxplot of top 50 enriched transcripts. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples

    Article Snippet: We compared 5 methods of library preparation for Illumina Next Generation sequencing: NuGEN Ovation RNA-Seq system V2 Kit, TaKaRa SMARTer Stranded Total RNA-Seq Kit, TaKaRa SMART-Seq v4 Ultra Low Input RNA Kit, Illumina TruSeq RNA Library Prep Kit v2 and NEBNext® Ultra™ Directional RNA Library Prep Kit using slightly modified protocols each with 4 ng of total RNA.

    Techniques:

    Distribution of normalized mean expression of the first (last) 100 bases of transcripts (in 5′- > 3′-orientation). X axis represents the 5′-3′ normalized position; Y axis represents normalized coverage. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuG samples; Green and grey: TruSeq samples. Solid: Input samples. Dotted: Ribo-IP samples

    Journal: BMC Genomics

    Article Title: A comparative analysis of library prep approaches for sequencing low input translatome samples

    doi: 10.1186/s12864-018-5066-2

    Figure Lengend Snippet: Distribution of normalized mean expression of the first (last) 100 bases of transcripts (in 5′- > 3′-orientation). X axis represents the 5′-3′ normalized position; Y axis represents normalized coverage. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuG samples; Green and grey: TruSeq samples. Solid: Input samples. Dotted: Ribo-IP samples

    Article Snippet: We compared 5 methods of library preparation for Illumina Next Generation sequencing: NuGEN Ovation RNA-Seq system V2 Kit, TaKaRa SMARTer Stranded Total RNA-Seq Kit, TaKaRa SMART-Seq v4 Ultra Low Input RNA Kit, Illumina TruSeq RNA Library Prep Kit v2 and NEBNext® Ultra™ Directional RNA Library Prep Kit using slightly modified protocols each with 4 ng of total RNA.

    Techniques: Expressing

    KMT2D loss leads to reduced keratinocyte proliferation and a broad loss of epithelial development and adhesion genes. ( A ) KMT2D mRNA expression is significantly reduced in both NHEKs ( P = 0.0036) and HaCaTs ( P = 0.0312) treated with shKMT2D as determined by RNA-seq. ( B ) shKMT2D keratinocytes display reduced levels of KMT2D but normal-appearing nuclear morphology. ( C ) shKMT2D keratinocytes display reduced proliferation in comparison with shSC keratinocytes. ( D ) Representative genes from shKMTD NHEKs that are significantly reduced in expression along with corresponding adjusted P -values. ( E ) Gene ontology (GO) analysis of the 134 common genes lost with shKMT2D treatment between both shKMT2D NHEKs and HaCaTs demonstrates that genes involved in key epithelial signaling pathways ( RARG and VDR ), epithelial cell growth and morphogenesis, polarity, and adhesion are enriched among those genes with reduced expression. ( F ) shKMT2D keratinocytes display reduced expression of RARγ and VDR by IF (40×).

    Journal: Genes & Development

    Article Title: KMT2D regulates p63 target enhancers to coordinate epithelial homeostasis

    doi: 10.1101/gad.306241.117

    Figure Lengend Snippet: KMT2D loss leads to reduced keratinocyte proliferation and a broad loss of epithelial development and adhesion genes. ( A ) KMT2D mRNA expression is significantly reduced in both NHEKs ( P = 0.0036) and HaCaTs ( P = 0.0312) treated with shKMT2D as determined by RNA-seq. ( B ) shKMT2D keratinocytes display reduced levels of KMT2D but normal-appearing nuclear morphology. ( C ) shKMT2D keratinocytes display reduced proliferation in comparison with shSC keratinocytes. ( D ) Representative genes from shKMTD NHEKs that are significantly reduced in expression along with corresponding adjusted P -values. ( E ) Gene ontology (GO) analysis of the 134 common genes lost with shKMT2D treatment between both shKMT2D NHEKs and HaCaTs demonstrates that genes involved in key epithelial signaling pathways ( RARG and VDR ), epithelial cell growth and morphogenesis, polarity, and adhesion are enriched among those genes with reduced expression. ( F ) shKMT2D keratinocytes display reduced expression of RARγ and VDR by IF (40×).

    Article Snippet: All RNA-seq libraries were prepared using the NEBNext poly(A) mRNA magnetic isolation module followed by NEBNext Ultra Directional RNA library preparation kit for Illumina (both from New England Biolabs).

    Techniques: Expressing, RNA Sequencing Assay

    EZH2 triggers cleavage of B2 RNA in vitro A) B2 sub-family consensus sequences. B) Incubation (22°C, 13h) of B2 RNA (200 nM) with EZH2 (25 nM) results in B2 cleavage. C) Incubation with 25 nM control proteins, GST and EED, does not result in significant cutting (22°C, 13h). D) Cleaved RNA fragments (asterisks) are subjected to deep sequencing (x-axis: start coordinates for the sequenced reads). E) Incubation of in vitro-transcribed RNAs (100 nM) with EZH2 (50 nM) results in cleavage only of B2. RNAs were mixed with EZH2 and incubated at 37°C or 4°C for 30 min. B2 was also incubated with FLAG peptide (50 nM) at 37°C as control. F) Kinetic analysis of B2 cleavage in the presence of EZH2. 25 nM EZH2 was incubated with 200 nM B2 RNA (37°C for 0–100 min.) and run on a 6% TBE-Urea-PAGE. G) Fraction of full-length B2 RNA at each time point from panel E (arrow) was plotted as a function of time (two independent experiments). Cleavage rate constants were then determined by a linear fit using the differential form of the rate equation for an irreversible, first-order reaction. The slope is the observed cleavage rate constant ( k obs ). H) Table of calculated ob k obs and RNA half-lives for B2 in the presence of various test proteins. I) Rate of B2 cleavage depends on the concentration of EZH2 protein. 50 nM B2 RNA is incubated with increasing concentrations of EZH2 in vitro (37°C, 20min) and run on a 6% TBE-Urea PAGE. J) Kinetic analysis showing that the rate of B2 cleavage depends on the concentration of EZH2 (two independent experiments). 200 nM B2 RNA is incubated with increasing EZH2 concentrations (25–500 nM) at 37°C and the amount of remaining full-length B2 RNA is plotted as a function of time. Cleavage rate constants were then determined by a linear fit using the differential form of the rate equation for an irreversible, first-order reaction. The slope approximated observed rate constant ( k obs ). K) k obs values from panel I are plotted as a function of EZH2 concentration. Arrowhead, full-length B2 RNA. Asterisks, cleaved B2 fragments. High R 2 , excellent fit of datapoints to the curve.

    Journal: Cell

    Article Title: Destabilization of B2 RNA by EZH2 activates the stress response

    doi: 10.1016/j.cell.2016.11.041

    Figure Lengend Snippet: EZH2 triggers cleavage of B2 RNA in vitro A) B2 sub-family consensus sequences. B) Incubation (22°C, 13h) of B2 RNA (200 nM) with EZH2 (25 nM) results in B2 cleavage. C) Incubation with 25 nM control proteins, GST and EED, does not result in significant cutting (22°C, 13h). D) Cleaved RNA fragments (asterisks) are subjected to deep sequencing (x-axis: start coordinates for the sequenced reads). E) Incubation of in vitro-transcribed RNAs (100 nM) with EZH2 (50 nM) results in cleavage only of B2. RNAs were mixed with EZH2 and incubated at 37°C or 4°C for 30 min. B2 was also incubated with FLAG peptide (50 nM) at 37°C as control. F) Kinetic analysis of B2 cleavage in the presence of EZH2. 25 nM EZH2 was incubated with 200 nM B2 RNA (37°C for 0–100 min.) and run on a 6% TBE-Urea-PAGE. G) Fraction of full-length B2 RNA at each time point from panel E (arrow) was plotted as a function of time (two independent experiments). Cleavage rate constants were then determined by a linear fit using the differential form of the rate equation for an irreversible, first-order reaction. The slope is the observed cleavage rate constant ( k obs ). H) Table of calculated ob k obs and RNA half-lives for B2 in the presence of various test proteins. I) Rate of B2 cleavage depends on the concentration of EZH2 protein. 50 nM B2 RNA is incubated with increasing concentrations of EZH2 in vitro (37°C, 20min) and run on a 6% TBE-Urea PAGE. J) Kinetic analysis showing that the rate of B2 cleavage depends on the concentration of EZH2 (two independent experiments). 200 nM B2 RNA is incubated with increasing EZH2 concentrations (25–500 nM) at 37°C and the amount of remaining full-length B2 RNA is plotted as a function of time. Cleavage rate constants were then determined by a linear fit using the differential form of the rate equation for an irreversible, first-order reaction. The slope approximated observed rate constant ( k obs ). K) k obs values from panel I are plotted as a function of EZH2 concentration. Arrowhead, full-length B2 RNA. Asterisks, cleaved B2 fragments. High R 2 , excellent fit of datapoints to the curve.

    Article Snippet: For sequencing of the in vitro B2 fragments no ribosomal depletion was applied For the longer RNAs we used the NEBNext Ultra directional RNA library kit (NEB) with an RNA fragmentation of 10 min at 95C and with the following modifications: First strand synthesis at 42C was done for 50 min and the End Prep of cDNA library was followed by an Ampure Beads selection of 1.8x and ligation of the adapters using the 5x quick ligation buffer and Quick T4 DNA ligase (NEB) for 30 min. Incubation with the USER enzyme was done before the PCR amplification for 30 min, followed by a double size selection of 0.5x–1x, while the final library was size selected using Ampure beads at a 1x sample-beads ratio.

    Techniques: In Vitro, Incubation, Sequencing, Polyacrylamide Gel Electrophoresis, Concentration Assay

    Schematic work flow of library preparation and sequencing using NEBNext Ultra Directional RNA Library Preparation kit.

    Journal: Data in Brief

    Article Title: Transcriptomic dataset reveals the molecular basis of genotypic variation in hexaploid wheat (T. aestivum L.) in response to Fe/Zn deficiency

    doi: 10.1016/j.dib.2020.105995

    Figure Lengend Snippet: Schematic work flow of library preparation and sequencing using NEBNext Ultra Directional RNA Library Preparation kit.

    Article Snippet: Library preparation and Illumina HiSeq sequencingRNA sequencing libraries were generated using Illumina-compatible NEBNext® Ultra™ Directional RNA Library Prep Kit (New England BioLabs, MA, USA) at Genotypic Technology Pvt.

    Techniques: Sequencing