ultra ii directional rna library prep kit  (New England Biolabs)


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    NEBNext Ultra II Directional RNA Library Prep Kit for Illumina
    Description:
    NEBNext Ultra II Directional RNA Library Prep Kit for Illumina 96 rxns
    Catalog Number:
    e7760l
    Price:
    3175
    Size:
    96 rxns
    Category:
    mRNA Template Preparation for PCR
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    New England Biolabs ultra ii directional rna library prep kit
    NEBNext Ultra II Directional RNA Library Prep Kit for Illumina
    NEBNext Ultra II Directional RNA Library Prep Kit for Illumina 96 rxns
    https://www.bioz.com/result/ultra ii directional rna library prep kit/product/New England Biolabs
    Average 99 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    ultra ii directional rna library prep kit - by Bioz Stars, 2020-04
    99/100 stars

    Images

    1) Product Images from "Sensory neuron lineage mapping and manipulation in the Drosophila olfactory system"

    Article Title: Sensory neuron lineage mapping and manipulation in the Drosophila olfactory system

    Journal: Nature Communications

    doi: 10.1038/s41467-019-08345-4

    Loss of Pnt results in Naa-to-Nab transformations in diverse sensillar subtypes. a A sensillum can contain up to four OSNs through differentiation of Naa (cyan), Nab (magenta), Nba (green), Nbb (yellow) terminal daughter cells originating from a single SOP lineage. b Representative images of RNA FISH for Or67d (magenta) in at1 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or67d-expressing OSNs are duplicated (arrow). A schematic of the proposed Naa-to-Nab fate transformation is shown on the right (color scheme as in ( a )). Scale bar = 2 µm. The open circles in this and other schematics represent OSN precursors that have undergone apoptosis. c Representative images of RNA FISH for Or85a (magenta) and Or59b (green) in ab2 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or85a OSNs (Nab) are duplicated (arrow), while Or59b OSNs (Nba) are unaffected. d Representative images of RNA FISH for Or85b (magenta) and Or22a (green) in ab3 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or85b OSNs (Nab) are duplicated (arrow), while Or22a OSNs (Nba) are unaffected. e Representative images of RNA FISH for Or92a (magenta) and Or10a (cyan) in ab1 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or92a OSNs (Nab) are duplicated (arrow), while Or10a OSNs (Naa) are lost. f Top: theoretical ratios of OSN types in 2-, 3- and 4-neuron sensilla in control and pnt RNAi antennae, assuming Naa-to-Nab fate transformation (i.e. loss of Naa OSNs, and duplication of Nab OSNs). Bottom: experimentally determined OSN ratios in all sensilla in pnt RNAi antennae using as a proxy the normalized ratios of olfactory receptor mRNA expression from antennal transcriptomes (see Supplementary Fig. 6e ). In ab10, Or49a is reported to be coexpressed with Or85f 13 , but transcript levels for this gene were below the cut-off applied during the analysis of these RNA-seq datasets
    Figure Legend Snippet: Loss of Pnt results in Naa-to-Nab transformations in diverse sensillar subtypes. a A sensillum can contain up to four OSNs through differentiation of Naa (cyan), Nab (magenta), Nba (green), Nbb (yellow) terminal daughter cells originating from a single SOP lineage. b Representative images of RNA FISH for Or67d (magenta) in at1 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or67d-expressing OSNs are duplicated (arrow). A schematic of the proposed Naa-to-Nab fate transformation is shown on the right (color scheme as in ( a )). Scale bar = 2 µm. The open circles in this and other schematics represent OSN precursors that have undergone apoptosis. c Representative images of RNA FISH for Or85a (magenta) and Or59b (green) in ab2 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or85a OSNs (Nab) are duplicated (arrow), while Or59b OSNs (Nba) are unaffected. d Representative images of RNA FISH for Or85b (magenta) and Or22a (green) in ab3 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or85b OSNs (Nab) are duplicated (arrow), while Or22a OSNs (Nba) are unaffected. e Representative images of RNA FISH for Or92a (magenta) and Or10a (cyan) in ab1 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or92a OSNs (Nab) are duplicated (arrow), while Or10a OSNs (Naa) are lost. f Top: theoretical ratios of OSN types in 2-, 3- and 4-neuron sensilla in control and pnt RNAi antennae, assuming Naa-to-Nab fate transformation (i.e. loss of Naa OSNs, and duplication of Nab OSNs). Bottom: experimentally determined OSN ratios in all sensilla in pnt RNAi antennae using as a proxy the normalized ratios of olfactory receptor mRNA expression from antennal transcriptomes (see Supplementary Fig. 6e ). In ab10, Or49a is reported to be coexpressed with Or85f 13 , but transcript levels for this gene were below the cut-off applied during the analysis of these RNA-seq datasets

    Techniques Used: Fluorescence In Situ Hybridization, Expressing, Transformation Assay, RNA Sequencing Assay

    An OSN lineage-specific driver. a Top row: developmental expression of the nonimmortalized GMR82D08-GAL4 (hereafter, at1 driver) using a myr:GFP reporter (green) in the antennal disc SOPs (region marked by α-Dac (blue)) during late larval/early pupal stages. Bottom row: the at1 driver is expressed in the daughter cells of these SOPs in the developing pupal antenna but progressively loses its expression from 20 h APF as OSNs differentiate (visualized with the neuronal marker α-Elav (magenta)). Scale bar = 20 µm in this and other panels. b Immortalization of the at1 driver reveals labeling of clusters of cells in the adult antenna by an rCD2:GFP reporter (green). RNA fluorescence in situ hybridization demonstrates that a single cell within each cluster (arrowheads in the inset images) expresses Or67d mRNA (magenta). c Representative example of a single sensillum in the adult antenna labeled by the immortalized at1 driver, viewed at three focal planes. There is a single Or67d mRNA-positive OSN (cell 1, arrowhead), flanked by four non-neuronal support cells (cells 2–5). d Sensilla cells labeled by the immortalized at1 driver lineage (α-GFP; green) also express Lush (magenta), an odorant binding protein unique to trichoid sensilla support cells 72
    Figure Legend Snippet: An OSN lineage-specific driver. a Top row: developmental expression of the nonimmortalized GMR82D08-GAL4 (hereafter, at1 driver) using a myr:GFP reporter (green) in the antennal disc SOPs (region marked by α-Dac (blue)) during late larval/early pupal stages. Bottom row: the at1 driver is expressed in the daughter cells of these SOPs in the developing pupal antenna but progressively loses its expression from 20 h APF as OSNs differentiate (visualized with the neuronal marker α-Elav (magenta)). Scale bar = 20 µm in this and other panels. b Immortalization of the at1 driver reveals labeling of clusters of cells in the adult antenna by an rCD2:GFP reporter (green). RNA fluorescence in situ hybridization demonstrates that a single cell within each cluster (arrowheads in the inset images) expresses Or67d mRNA (magenta). c Representative example of a single sensillum in the adult antenna labeled by the immortalized at1 driver, viewed at three focal planes. There is a single Or67d mRNA-positive OSN (cell 1, arrowhead), flanked by four non-neuronal support cells (cells 2–5). d Sensilla cells labeled by the immortalized at1 driver lineage (α-GFP; green) also express Lush (magenta), an odorant binding protein unique to trichoid sensilla support cells 72

    Techniques Used: Expressing, Marker, Labeling, Fluorescence, In Situ Hybridization, Binding Assay

    2) Product Images from "Sensory neuron lineage mapping and manipulation in the Drosophila olfactory system"

    Article Title: Sensory neuron lineage mapping and manipulation in the Drosophila olfactory system

    Journal: Nature Communications

    doi: 10.1038/s41467-019-08345-4

    An OSN lineage-specific driver. a Top row: developmental expression of the nonimmortalized GMR82D08-GAL4 (hereafter, at1 driver) using a myr:GFP reporter (green) in the antennal disc SOPs (region marked by α-Dac (blue)) during late larval/early pupal stages. Bottom row: the at1 driver is expressed in the daughter cells of these SOPs in the developing pupal antenna but progressively loses its expression from 20 h APF as OSNs differentiate (visualized with the neuronal marker α-Elav (magenta)). Scale bar = 20 µm in this and other panels. b Immortalization of the at1 driver reveals labeling of clusters of cells in the adult antenna by an rCD2:GFP reporter (green). RNA fluorescence in situ hybridization demonstrates that a single cell within each cluster (arrowheads in the inset images) expresses Or67d mRNA (magenta). c Representative example of a single sensillum in the adult antenna labeled by the immortalized at1 driver, viewed at three focal planes. There is a single Or67d mRNA-positive OSN (cell 1, arrowhead), flanked by four non-neuronal support cells (cells 2–5). d
    Figure Legend Snippet: An OSN lineage-specific driver. a Top row: developmental expression of the nonimmortalized GMR82D08-GAL4 (hereafter, at1 driver) using a myr:GFP reporter (green) in the antennal disc SOPs (region marked by α-Dac (blue)) during late larval/early pupal stages. Bottom row: the at1 driver is expressed in the daughter cells of these SOPs in the developing pupal antenna but progressively loses its expression from 20 h APF as OSNs differentiate (visualized with the neuronal marker α-Elav (magenta)). Scale bar = 20 µm in this and other panels. b Immortalization of the at1 driver reveals labeling of clusters of cells in the adult antenna by an rCD2:GFP reporter (green). RNA fluorescence in situ hybridization demonstrates that a single cell within each cluster (arrowheads in the inset images) expresses Or67d mRNA (magenta). c Representative example of a single sensillum in the adult antenna labeled by the immortalized at1 driver, viewed at three focal planes. There is a single Or67d mRNA-positive OSN (cell 1, arrowhead), flanked by four non-neuronal support cells (cells 2–5). d

    Techniques Used: Expressing, Marker, Labeling, Fluorescence, In Situ Hybridization

    3) Product Images from "Testing of library preparation methods for transcriptome sequencing of real life glioblastoma and brain tissue specimens: A comparative study with special focus on long non-coding RNAs"

    Article Title: Testing of library preparation methods for transcriptome sequencing of real life glioblastoma and brain tissue specimens: A comparative study with special focus on long non-coding RNAs

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0211978

    Results from comparison of three library preparation kits using the Ensembl automatic annotation system. The number of hits for each transcript biotype from Ensembl database calculated for each group of libraries generated using the same library preparation kit is on y-axis. Only the most populous biotypes relevant to our current study were included. While the first biotype comprises the majority of the group of protein coding RNA, the rest (lincRNA, antisense and sense intronic RNA) is part of another large group of long non-coding RNA. After comparing all three kits, the highest number of hits for each biotype of interest was observed for NEBNext (NB) libraries; NEXTflex (NF) libraries came second and SENSE (LX) libraries third. These results hint at a lesser variability of library fragments in LX libraries.
    Figure Legend Snippet: Results from comparison of three library preparation kits using the Ensembl automatic annotation system. The number of hits for each transcript biotype from Ensembl database calculated for each group of libraries generated using the same library preparation kit is on y-axis. Only the most populous biotypes relevant to our current study were included. While the first biotype comprises the majority of the group of protein coding RNA, the rest (lincRNA, antisense and sense intronic RNA) is part of another large group of long non-coding RNA. After comparing all three kits, the highest number of hits for each biotype of interest was observed for NEBNext (NB) libraries; NEXTflex (NF) libraries came second and SENSE (LX) libraries third. These results hint at a lesser variability of library fragments in LX libraries.

    Techniques Used: Generated

    4) Product Images from "Transcriptome Analysis of Intrusively Growing Flax Fibers Isolated by Laser Microdissection"

    Article Title: Transcriptome Analysis of Intrusively Growing Flax Fibers Isolated by Laser Microdissection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-32869-2

    ( A ) Overrepresentation analysis of up- and down-regulated genes within functional gene classes defined by MapMan bins (MapMan, v3.6.0RC1, available online at http://mapman.gabipd.org/web/guest/mapman [accessed on 29 June 2018]) in iFIB sample as compared to APEX (iFIB/APEX). The data were subjected to a Wilcoxon test; resulting p-values were adjusted according to Benjamini and Hochberg in PageMan ( http://mapman.gabipd.org/pageman [accessed on 20 July 2018]), and the results are displayed in false color. Functional gene classes colored in red are significantly up-regulated, whereas ones colored in green are significantly down-regulated. PS, photosynthesis; TCA, tricarboxylic acid cycle; AA, amino acid; CHO, carbohydrate. ( B ) Verification of RNA-Seq expression through qRT-PCR, error bar shows the standard error of the mean.
    Figure Legend Snippet: ( A ) Overrepresentation analysis of up- and down-regulated genes within functional gene classes defined by MapMan bins (MapMan, v3.6.0RC1, available online at http://mapman.gabipd.org/web/guest/mapman [accessed on 29 June 2018]) in iFIB sample as compared to APEX (iFIB/APEX). The data were subjected to a Wilcoxon test; resulting p-values were adjusted according to Benjamini and Hochberg in PageMan ( http://mapman.gabipd.org/pageman [accessed on 20 July 2018]), and the results are displayed in false color. Functional gene classes colored in red are significantly up-regulated, whereas ones colored in green are significantly down-regulated. PS, photosynthesis; TCA, tricarboxylic acid cycle; AA, amino acid; CHO, carbohydrate. ( B ) Verification of RNA-Seq expression through qRT-PCR, error bar shows the standard error of the mean.

    Techniques Used: Functional Assay, RNA Sequencing Assay, Expressing, Quantitative RT-PCR

    Scheme of plant material collection to obtain APEX, iFIB, FIB samples for subsequent RNA-Seq analysis. Bundles of the intrusively growing fibers (sample iFIB) were isolated by laser microdissection from the longitudinal cryosections of 3rd cm from the stem apex. Sample APEX was collected as 2 mm of the uppermost stem together with leaf primordia. The described earlier 12 sample FIB (fibers isolated during tertiary cell wall deposition) was collected from the stem portion below the snap point (SP).
    Figure Legend Snippet: Scheme of plant material collection to obtain APEX, iFIB, FIB samples for subsequent RNA-Seq analysis. Bundles of the intrusively growing fibers (sample iFIB) were isolated by laser microdissection from the longitudinal cryosections of 3rd cm from the stem apex. Sample APEX was collected as 2 mm of the uppermost stem together with leaf primordia. The described earlier 12 sample FIB (fibers isolated during tertiary cell wall deposition) was collected from the stem portion below the snap point (SP).

    Techniques Used: RNA Sequencing Assay, Isolation, Laser Capture Microdissection

    5) Product Images from "Intrusive Growth of Phloem Fibers in Flax Stem: Integrated Analysis of miRNA and mRNA Expression Profiles"

    Article Title: Intrusive Growth of Phloem Fibers in Flax Stem: Integrated Analysis of miRNA and mRNA Expression Profiles

    Journal: Plants

    doi: 10.3390/plants8020047

    A scheme of plant material collection to obtain iFIBa, iFIBb, cPAR, sXYL and tFIB samples for subsequent RNA-Seq and miRNA-Seq analysis. Bundles of intrusively (i) growing fibers (the samples iFIBa and iFIBb) were isolated from longitudinal cryosections of the stem part a (0.3–0.8 cm from the stem apex) and b (0.8–2.5 cm from the stem apex) by laser microdissection. Cortex (c) parenchyma (cPAR) was isolated from longitudinal cryosections of the stem parts a and b by laser microdissection, and these two tissue fractions were combined. Fibers depositing tertiary (t) cell walls (tFIB) and xylem enriched in secondary (s) cell walls (sXYL) were sampled 1 cm below the snap point. E—epidermis, F—fiber bundles, L—leaf, P—parenchyma, and X—xylem; Bar—200 µm.
    Figure Legend Snippet: A scheme of plant material collection to obtain iFIBa, iFIBb, cPAR, sXYL and tFIB samples for subsequent RNA-Seq and miRNA-Seq analysis. Bundles of intrusively (i) growing fibers (the samples iFIBa and iFIBb) were isolated from longitudinal cryosections of the stem part a (0.3–0.8 cm from the stem apex) and b (0.8–2.5 cm from the stem apex) by laser microdissection. Cortex (c) parenchyma (cPAR) was isolated from longitudinal cryosections of the stem parts a and b by laser microdissection, and these two tissue fractions were combined. Fibers depositing tertiary (t) cell walls (tFIB) and xylem enriched in secondary (s) cell walls (sXYL) were sampled 1 cm below the snap point. E—epidermis, F—fiber bundles, L—leaf, P—parenchyma, and X—xylem; Bar—200 µm.

    Techniques Used: RNA Sequencing Assay, Isolation, Laser Capture Microdissection

    6) Product Images from "Isolation and genome-wide characterization of cellular DNA:RNA triplex structures"

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1305

    NEAT1 forms triplexes at numerous genomic sites. ( A ) NEAT1 profiles in TriplexRNA-seq (DNA-IP) (red) and nuclear RNA (blue) from HeLa S3 and U2OS cells with shaded TFR1 and TFR2. Minus (-) and plus (+) strands are shown. The position and sequence of NEAT1-TFR1 and -TFR2 are shown below. ( B ) EMSAs using 10 or 100 pmol of synthetic NEAT1 versions comprising TFR1 (40 or 52 nt) or TFR2 incubated with 0.25 pmol of double–stranded 32 P-labeled oligonucleotides which harbor sequences of NEAT1 target genes predicted from CHART-seq ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control, RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( C ) Schematic depiction of the TFR-based capture assay. Biotinylated RNA oligos covering NEAT1-TFR1 and NEAT1-TFR2 were used to capture genomic DNA. ( D ) MEME motif analysis identifying consensus motifs in DNA captured by NEAT1-TFR1 (399 of top 500 peaks) and by NEAT1-TFR2 (500 of top 500 peaks ranked by peak P -value). Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( E ) TDF analysis of the triplex-forming potential of NEAT1-TFR1 and NEAT1-TFR2 RNAs with top 500 TFR-associated and control DNA peaks (ranked by peak P -value) compared to 500 randomized regions ( N = 1000, colored grey). P -values were obtained from one-tailed Mann–Whitney test. ( F ) Scheme presenting antisense oligo (ASO)-based capture of NEAT1-associated DNA. ( G ) Consensus motif in NEAT1-associated DNA sites (314 of top 500 peaks ranked by peak P -value). ( H ) TDF analysis predicting the triplex-forming potential of NEAT1 on ASO-captured DNA regions. Significant TFRs along NEAT1 are shown in orange, the number of target sites (DBS) for each TFR in purple. For TFR- and ASO-based capture assays nucleic acids isolated from HeLa S3 chromatin were used.
    Figure Legend Snippet: NEAT1 forms triplexes at numerous genomic sites. ( A ) NEAT1 profiles in TriplexRNA-seq (DNA-IP) (red) and nuclear RNA (blue) from HeLa S3 and U2OS cells with shaded TFR1 and TFR2. Minus (-) and plus (+) strands are shown. The position and sequence of NEAT1-TFR1 and -TFR2 are shown below. ( B ) EMSAs using 10 or 100 pmol of synthetic NEAT1 versions comprising TFR1 (40 or 52 nt) or TFR2 incubated with 0.25 pmol of double–stranded 32 P-labeled oligonucleotides which harbor sequences of NEAT1 target genes predicted from CHART-seq ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control, RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( C ) Schematic depiction of the TFR-based capture assay. Biotinylated RNA oligos covering NEAT1-TFR1 and NEAT1-TFR2 were used to capture genomic DNA. ( D ) MEME motif analysis identifying consensus motifs in DNA captured by NEAT1-TFR1 (399 of top 500 peaks) and by NEAT1-TFR2 (500 of top 500 peaks ranked by peak P -value). Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( E ) TDF analysis of the triplex-forming potential of NEAT1-TFR1 and NEAT1-TFR2 RNAs with top 500 TFR-associated and control DNA peaks (ranked by peak P -value) compared to 500 randomized regions ( N = 1000, colored grey). P -values were obtained from one-tailed Mann–Whitney test. ( F ) Scheme presenting antisense oligo (ASO)-based capture of NEAT1-associated DNA. ( G ) Consensus motif in NEAT1-associated DNA sites (314 of top 500 peaks ranked by peak P -value). ( H ) TDF analysis predicting the triplex-forming potential of NEAT1 on ASO-captured DNA regions. Significant TFRs along NEAT1 are shown in orange, the number of target sites (DBS) for each TFR in purple. For TFR- and ASO-based capture assays nucleic acids isolated from HeLa S3 chromatin were used.

    Techniques Used: Sequencing, Incubation, Labeling, One-tailed Test, MANN-WHITNEY, Allele-specific Oligonucleotide, Isolation

    Validation of triplex-forming RNA and DNAs. ( A ) TDF analysis predicting the potential of top 1000 enriched TriplexRNA (DNA-IP) regions (ranked by peak P -value) to bind to active promoters defined by ChromHMM. Number of TFRs in RNA (per kilobase of RNA, left) and the number of putative DBSs at promoters (per kilobase of RNA, right) are shown. Boxplot borders are defined by the 1st and 3rd quantiles of the distributions, the middle line corresponds to the median value. The top whisker denotes the maximum value within the third quartile plus 1.5 times the interquartile range (bottom whisker is defined analogously). Dark gray dots represent outliers with values higher or lower than whiskers. Further box plots are based on the same definitions. ( B ) Motif analysis of triplexes formed between TriplexRNA (DNA-IP) and active promoters. The diagram depicts the fraction of antiparallel and parallel triplexes with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( C ) TDF analysis comparing the triplex-forming potential of top 2000 TriplexDNA-seq regions with top 1000 TriplexRNA (DNA-IP) (ranked by peak P -value). The number of putative DBSs (per kilobase of RNA) is shown. ( D ) Motif analysis of predicted triplexes formed between TriplexRNAs (DNA-IP) and TriplexDNA. The diagram depicts the fraction of antiparallel and parallel triplexes, with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( E ) Box plot classifying triplex interactions between TriplexRNAs (DNA-IP) and TriplexDNA-seq regions as cis ( > 10 kb in the same chromosome) and trans (at different chromosomes) interactions, excluding underrepresented local interactions (within 10 kb distance). ( F ) EMSAs using 10 or 100 pmol of synthetic TriplexRNAs and 0.25 pmol of double–stranded 32 P-labeled oligonucleotides comprising target regions from TriplexDNA ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control (C), RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). TriplexRNA-seq and TriplexDNA-seq data are from HeLa S3 cells. Adjusted P -values
    Figure Legend Snippet: Validation of triplex-forming RNA and DNAs. ( A ) TDF analysis predicting the potential of top 1000 enriched TriplexRNA (DNA-IP) regions (ranked by peak P -value) to bind to active promoters defined by ChromHMM. Number of TFRs in RNA (per kilobase of RNA, left) and the number of putative DBSs at promoters (per kilobase of RNA, right) are shown. Boxplot borders are defined by the 1st and 3rd quantiles of the distributions, the middle line corresponds to the median value. The top whisker denotes the maximum value within the third quartile plus 1.5 times the interquartile range (bottom whisker is defined analogously). Dark gray dots represent outliers with values higher or lower than whiskers. Further box plots are based on the same definitions. ( B ) Motif analysis of triplexes formed between TriplexRNA (DNA-IP) and active promoters. The diagram depicts the fraction of antiparallel and parallel triplexes with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( C ) TDF analysis comparing the triplex-forming potential of top 2000 TriplexDNA-seq regions with top 1000 TriplexRNA (DNA-IP) (ranked by peak P -value). The number of putative DBSs (per kilobase of RNA) is shown. ( D ) Motif analysis of predicted triplexes formed between TriplexRNAs (DNA-IP) and TriplexDNA. The diagram depicts the fraction of antiparallel and parallel triplexes, with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( E ) Box plot classifying triplex interactions between TriplexRNAs (DNA-IP) and TriplexDNA-seq regions as cis ( > 10 kb in the same chromosome) and trans (at different chromosomes) interactions, excluding underrepresented local interactions (within 10 kb distance). ( F ) EMSAs using 10 or 100 pmol of synthetic TriplexRNAs and 0.25 pmol of double–stranded 32 P-labeled oligonucleotides comprising target regions from TriplexDNA ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control (C), RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). TriplexRNA-seq and TriplexDNA-seq data are from HeLa S3 cells. Adjusted P -values

    Techniques Used: Whisker Assay, Labeling

    7) Product Images from "Antiviral potential of human IFN-α subtypes against influenza A H3N2 infection in human lung explants reveals subtype-specific activities"

    Article Title: Antiviral potential of human IFN-α subtypes against influenza A H3N2 infection in human lung explants reveals subtype-specific activities

    Journal: Emerging Microbes & Infections

    doi: 10.1080/22221751.2019.1698271

    Innate immune response to H3N2 IAV infection in human lung explants. Tissue was infected with 1 × 10 5 PFU/mL of H3N2 IAV or mock-infected. (A) Total RNA was isolated 24 hpi for RNA-sequencing. mRNA expression levels are depicted as mean log 2 fold change compared to mock-infected tissue. n = 5 independent donors. Multiple testing with BH correction. (B) mRNAs of individual ISGs and cytokines were analysed by qRT-PCR. GAPDH was used as the housekeeping gene. (C) Secreted cytokines were analysed in the supernatants at 24 hpi. Individual results of infected and mock-infected lung tissues are derived from the same donor. Bars represent mean (±SEM). * p
    Figure Legend Snippet: Innate immune response to H3N2 IAV infection in human lung explants. Tissue was infected with 1 × 10 5 PFU/mL of H3N2 IAV or mock-infected. (A) Total RNA was isolated 24 hpi for RNA-sequencing. mRNA expression levels are depicted as mean log 2 fold change compared to mock-infected tissue. n = 5 independent donors. Multiple testing with BH correction. (B) mRNAs of individual ISGs and cytokines were analysed by qRT-PCR. GAPDH was used as the housekeeping gene. (C) Secreted cytokines were analysed in the supernatants at 24 hpi. Individual results of infected and mock-infected lung tissues are derived from the same donor. Bars represent mean (±SEM). * p

    Techniques Used: Infection, Isolation, RNA Sequencing Assay, Expressing, Quantitative RT-PCR, Derivative Assay

    Induction of IFN-α subtypes mRNA levels by H3N2 IAV infection in human lung explants. Total RNA was isolated 24 hpi for RNA-sequencing. mRNA levels are depicted as mean log 2 fold change, compared to mock-infected tissue from the same donor. n = 5 independent donors. * p
    Figure Legend Snippet: Induction of IFN-α subtypes mRNA levels by H3N2 IAV infection in human lung explants. Total RNA was isolated 24 hpi for RNA-sequencing. mRNA levels are depicted as mean log 2 fold change, compared to mock-infected tissue from the same donor. n = 5 independent donors. * p

    Techniques Used: Infection, Isolation, RNA Sequencing Assay

    8) Product Images from "Clonotypic heterogeneity in cutaneous T-cell lymphoma (mycosis fungoides) revealed by comprehensive whole-exome sequencing"

    Article Title: Clonotypic heterogeneity in cutaneous T-cell lymphoma (mycosis fungoides) revealed by comprehensive whole-exome sequencing

    Journal: Blood Advances

    doi: 10.1182/bloodadvances.2018027482

    Schematic representation of sample collection, processing, and TCR sequencing. Four-millimeter punch biopsies were collected from early lesions (plaques; red circles) or tumors (green squares) in 27 patients with MF. Biopsies were cryosectioned and laser microdissected to capture tumor cells that were pooled together. Original magnification ×10; hematoxylin and eosin staining. DNA and RNA were isolated simultaneously from the microdissected material and processed for WES and WTS. WTS data are available only for samples MF4_2T, MF4_3P, MF5_1T, MF5_2P, MF7_1T, MF7_2P, MF11T, MF11_1P, MF19_1T, and MF19_2P and a pool of normal CD4 + lymphocytes (data not shown). The gene sequence is indicated in green, the adapter sequence is indicated in red, and the index sequence is indicated in blue.
    Figure Legend Snippet: Schematic representation of sample collection, processing, and TCR sequencing. Four-millimeter punch biopsies were collected from early lesions (plaques; red circles) or tumors (green squares) in 27 patients with MF. Biopsies were cryosectioned and laser microdissected to capture tumor cells that were pooled together. Original magnification ×10; hematoxylin and eosin staining. DNA and RNA were isolated simultaneously from the microdissected material and processed for WES and WTS. WTS data are available only for samples MF4_2T, MF4_3P, MF5_1T, MF5_2P, MF7_1T, MF7_2P, MF11T, MF11_1P, MF19_1T, and MF19_2P and a pool of normal CD4 + lymphocytes (data not shown). The gene sequence is indicated in green, the adapter sequence is indicated in red, and the index sequence is indicated in blue.

    Techniques Used: Sequencing, Staining, Isolation

    Related Articles

    Multiplex Assay:

    Article Title: Intrusive Growth of Phloem Fibers in Flax Stem: Integrated Analysis of miRNA and mRNA Expression Profiles
    Article Snippet: Total RNA of each sample was used to prepare both RNA-library and small RNA library. cDNA libraries were prepared from the total RNA of iFIBa, iFIBb, cPAR, tFIB, and sXYL samples (up to 1 µg) with a NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA) after selective depletion of ribosomal RNAs using a Ribo-Zero rRNA Removal Kit (Plant) (Illumina, San Diego, CA, USA), according to the manufacturer’s instructions. .. Libraries of small RNA were prepared with a NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s protocol.

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures
    Article Snippet: .. Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit and NEBNext Multiplex Oligos for Illumina (NEB). ..

    Selection:

    Article Title: Intrusive Growth of Phloem Fibers in Flax Stem: Integrated Analysis of miRNA and mRNA Expression Profiles
    Article Snippet: Total RNA of each sample was used to prepare both RNA-library and small RNA library. cDNA libraries were prepared from the total RNA of iFIBa, iFIBb, cPAR, tFIB, and sXYL samples (up to 1 µg) with a NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA) after selective depletion of ribosomal RNAs using a Ribo-Zero rRNA Removal Kit (Plant) (Illumina, San Diego, CA, USA), according to the manufacturer’s instructions. .. Further size selection was performed using Agencourt AMPure XP (Beckman Coulter, Indianapolis, Indiana, USA) to select for 143–149 bp fragments.

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures
    Article Snippet: Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit and NEBNext Multiplex Oligos for Illumina (NEB). .. Libraries from HeLa S3 cells were from independent experiments monitoring DNA-associated RNA from DNA-IP (N=3) and SPRI-size selection (N=4) as well as chromatin-associated RNA (N=4) and nuclear RNA (N=2).

    RNA Sequencing Assay:

    Article Title: Antiviral potential of human IFN-α subtypes against influenza A H3N2 infection in human lung explants reveals subtype-specific activities
    Article Snippet: .. The RNA sequencing library was generated from 500 ng total RNA using Dynabeads mRNA DIRECT Micro Purification Kit (Thermo Fisher Scientific, USA), for mRNA purification, followed by NEBNext Ultra II Directional RNA Library Prep Kit (New England BioLabs, USA), according to manufacturer’s protocols. .. The libraries were sequenced on Illumina NovaSeq 6000 using NovaSeq 6000 S1 Reagent Kit (200 and 300 cycles, paired end runs) with an average of 7.9 × 107 reads per sample.

    Article Title: Sensory neuron lineage mapping and manipulation in the Drosophila olfactory system
    Article Snippet: .. RNA-seq libraries were prepared from the mRNA using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). .. Cluster generation was performed with the resulting libraries using the Illumina TruSeq SR Cluster Kit v4 reagents and sequenced on the Illumina HiSeq 2500 using TruSeq SBS Kit v4 reagents (Illumina).

    Article Title: Testing of library preparation methods for transcriptome sequencing of real life glioblastoma and brain tissue specimens: A comparative study with special focus on long non-coding RNAs
    Article Snippet: .. In the present study, we used GBM and non-tumor brain tissue specimens and compared three different commercial library preparation kits, namely NEXTflex Rapid Directional qRNA-Seq Kit (Bioo Scientific), SENSE Total RNA-Seq Library Prep Kit (Lexogen) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB). .. Libraries generated using SENSE kit were characterized by the most normal distribution of normalized average GC content, the least amount of over-represented sequences and the percentage of ribosomal RNA reads (0.3–1.5%) and highest numbers of uniquely mapped reads and reads aligning to coding regions.

    Article Title: Transcriptome engineering with RNA-targeting Type VI-D CRISPR effectors
    Article Snippet: Paragraph title: RNA-seq library preparation and sequencing ... Stranded mRNA libraries were prepared using the NEBNext II Ultra Directional RNA Library Prep Kit from New England Biolabs (Cat# E7760S) and sequenced on an Illumina NextSeq500 with 42 nt paired end reads.

    Article Title: Transcriptome Analysis of Intrusively Growing Flax Fibers Isolated by Laser Microdissection
    Article Snippet: .. To produce normalized cDNA libraries, followed by sequencing using Illumina MiSeq with single-end 75 bp reads. cDNA libraries from total RNA of iFIB samples (up to 1 µg) were prepared with NEBNext Ultra II Directional RNA Library Prep Kit after selective depletion of ribosomal RNA using RiboMinus™ Plant Kit for RNA-Seq according to the manufacturer’s instructions. .. Sequence was performed on HiSeq2500 with 60 bp single-end reads.

    Article Title: JASPer controls interphase histone H3S10 phosphorylation by chromosomal kinase JIL-1 in Drosophila
    Article Snippet: Paragraph title: RNA-seq ... Library preparation was done according to the manufacturer’s instructions with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, E7760) and analyzed with 2100 Bioanalyzer with DNA 1000 kit (Agilent).

    RNA HS Assay:

    Article Title: Clonotypic heterogeneity in cutaneous T-cell lymphoma (mycosis fungoides) revealed by comprehensive whole-exome sequencing
    Article Snippet: Ten nanograms of total RNA, quantified using a Qubit RNA HS Assay Kit (Q32852; Thermo Fisher Scientific), was used for ribosomal RNA depletion using a NEBNext rRNA Depletion Kit (catalog number E6310; New England Biolabs). .. Ribosomal RNA–depleted samples were used for complementary DNA (cDNA) synthesis, and the library was built using a NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (catalog number E7760; New England Biolabs).

    Ligation:

    Article Title: R-Loops Promote Antisense Transcription across the Mammalian Genome
    Article Snippet: The resulting 5′P RNA enables single stranded RNA ligation of adaptor rp5: (5′CTTTCCCTACACGACGCTCTTCCGATrCrUrNrNrNrNrNrNrNrN-3′) with T4 RNA ligase (NEB M0204S). .. These RNA libraries were prepared with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB) according to the manufacturer’s guidelines.

    Isolation:

    Article Title: Intrusive Growth of Phloem Fibers in Flax Stem: Integrated Analysis of miRNA and mRNA Expression Profiles
    Article Snippet: Paragraph title: 4.3. RNA Isolation, Library Preparation and Sequencing ... Total RNA of each sample was used to prepare both RNA-library and small RNA library. cDNA libraries were prepared from the total RNA of iFIBa, iFIBb, cPAR, tFIB, and sXYL samples (up to 1 µg) with a NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA) after selective depletion of ribosomal RNAs using a Ribo-Zero rRNA Removal Kit (Plant) (Illumina, San Diego, CA, USA), according to the manufacturer’s instructions.

    Article Title: Sensory neuron lineage mapping and manipulation in the Drosophila olfactory system
    Article Snippet: From 100 ng total RNA, mRNA was isolated with the NEBNext Poly(A) mRNA Magnetic Isolation Module. .. RNA-seq libraries were prepared from the mRNA using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs).

    Article Title: Transcriptome Analysis of Intrusively Growing Flax Fibers Isolated by Laser Microdissection
    Article Snippet: Total RNA from the APEX (20 plants per sample) was isolated using Trizol extraction method combined with RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. .. To produce normalized cDNA libraries, followed by sequencing using Illumina MiSeq with single-end 75 bp reads. cDNA libraries from total RNA of iFIB samples (up to 1 µg) were prepared with NEBNext Ultra II Directional RNA Library Prep Kit after selective depletion of ribosomal RNA using RiboMinus™ Plant Kit for RNA-Seq according to the manufacturer’s instructions.

    Next-Generation Sequencing:

    Article Title: Intrusive Growth of Phloem Fibers in Flax Stem: Integrated Analysis of miRNA and mRNA Expression Profiles
    Article Snippet: The RNA integrity index (RIN) was 7–8.4, which provides sufficient integrity for NGS. .. Total RNA of each sample was used to prepare both RNA-library and small RNA library. cDNA libraries were prepared from the total RNA of iFIBa, iFIBb, cPAR, tFIB, and sXYL samples (up to 1 µg) with a NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA) after selective depletion of ribosomal RNAs using a Ribo-Zero rRNA Removal Kit (Plant) (Illumina, San Diego, CA, USA), according to the manufacturer’s instructions.

    Article Title: Testing of library preparation methods for transcriptome sequencing of real life glioblastoma and brain tissue specimens: A comparative study with special focus on long non-coding RNAs
    Article Snippet: Since the discovery of long non-coding transcripts and their multiple regulatory functions, new array of methods including NGS gave rise to a whole new field of genomics and epigentics which focuses on these molecules and their implication in the context of pathophysiological processes. .. Out of the three library preparation kits we tested, NEBNext Ultra II Directional RNA Library Prep for Illumina kit performed best with regard to relatively even transcript coverage, low rates of PCR duplicates and the highest number of hits for biotypes of interest in Ensembl database.

    Article Title: Transcriptome Analysis of Intrusively Growing Flax Fibers Isolated by Laser Microdissection
    Article Snippet: The RNA integrity index (RIN) was more than 6.2, which corresponds to the quality of RNA for further analysis by methods NGS. .. To produce normalized cDNA libraries, followed by sequencing using Illumina MiSeq with single-end 75 bp reads. cDNA libraries from total RNA of iFIB samples (up to 1 µg) were prepared with NEBNext Ultra II Directional RNA Library Prep Kit after selective depletion of ribosomal RNA using RiboMinus™ Plant Kit for RNA-Seq according to the manufacturer’s instructions.

    Transfection:

    Article Title: Transcriptome engineering with RNA-targeting Type VI-D CRISPR effectors
    Article Snippet: 48h after transfection, total RNA was extracted from 293FT cells using the RNeasy Plus Mini kit from Qiagen. .. Stranded mRNA libraries were prepared using the NEBNext II Ultra Directional RNA Library Prep Kit from New England Biolabs (Cat# E7760S) and sequenced on an Illumina NextSeq500 with 42 nt paired end reads.

    Purification:

    Article Title: Antiviral potential of human IFN-α subtypes against influenza A H3N2 infection in human lung explants reveals subtype-specific activities
    Article Snippet: .. The RNA sequencing library was generated from 500 ng total RNA using Dynabeads mRNA DIRECT Micro Purification Kit (Thermo Fisher Scientific, USA), for mRNA purification, followed by NEBNext Ultra II Directional RNA Library Prep Kit (New England BioLabs, USA), according to manufacturer’s protocols. .. The libraries were sequenced on Illumina NovaSeq 6000 using NovaSeq 6000 S1 Reagent Kit (200 and 300 cycles, paired end runs) with an average of 7.9 × 107 reads per sample.

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures
    Article Snippet: Preparation of libraries RNA samples were treated with 2 U of DNase I for 20 min at room temperature, purified with TRI reagent, and rRNA was depleted with the NEBNext rRNA depletion kit (NEB). .. Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit and NEBNext Multiplex Oligos for Illumina (NEB).

    Article Title: JASPer controls interphase histone H3S10 phosphorylation by chromosomal kinase JIL-1 in Drosophila
    Article Snippet: Afterwards, 1 µg of purified total RNA’s was used for rRNA depletion using Ribo-Zero Gold rRNA Removal Kit (Illumnia, MRZG 12324) or NEBNext rRNA Depletion Kit (NEB, E6310). .. Library preparation was done according to the manufacturer’s instructions with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, E7760) and analyzed with 2100 Bioanalyzer with DNA 1000 kit (Agilent).

    Sequencing:

    Article Title: Intrusive Growth of Phloem Fibers in Flax Stem: Integrated Analysis of miRNA and mRNA Expression Profiles
    Article Snippet: Paragraph title: 4.3. RNA Isolation, Library Preparation and Sequencing ... Total RNA of each sample was used to prepare both RNA-library and small RNA library. cDNA libraries were prepared from the total RNA of iFIBa, iFIBb, cPAR, tFIB, and sXYL samples (up to 1 µg) with a NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA) after selective depletion of ribosomal RNAs using a Ribo-Zero rRNA Removal Kit (Plant) (Illumina, San Diego, CA, USA), according to the manufacturer’s instructions.

    Article Title: Testing of library preparation methods for transcriptome sequencing of real life glioblastoma and brain tissue specimens: A comparative study with special focus on long non-coding RNAs
    Article Snippet: Considering our findings, we conclude that choosing the most suitable approach to library construction for transcriptome sequencing with the aim of studying lncRNAs is highly important in order to retain valuable data on lowly expressed RNA species that could otherwise be permanently lost. .. Out of the three library preparation kits we tested, NEBNext Ultra II Directional RNA Library Prep for Illumina kit performed best with regard to relatively even transcript coverage, low rates of PCR duplicates and the highest number of hits for biotypes of interest in Ensembl database.

    Article Title: Sensory neuron lineage mapping and manipulation in the Drosophila olfactory system
    Article Snippet: Paragraph title: RNA library preparation and sequencing ... RNA-seq libraries were prepared from the mRNA using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs).

    Article Title: Clonotypic heterogeneity in cutaneous T-cell lymphoma (mycosis fungoides) revealed by comprehensive whole-exome sequencing
    Article Snippet: Paragraph title: Sample preparation for whole-transcriptome sequencing ... Ribosomal RNA–depleted samples were used for complementary DNA (cDNA) synthesis, and the library was built using a NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (catalog number E7760; New England Biolabs).

    Article Title: Testing of library preparation methods for transcriptome sequencing of real life glioblastoma and brain tissue specimens: A comparative study with special focus on long non-coding RNAs
    Article Snippet: To maximize the value of transcriptome sequencing, a proper protocol for library preparation from tissue-derived RNA needs to be found which would produce high quality transcriptome sequencing data and increase the number of detected lncRNAs. .. In the present study, we used GBM and non-tumor brain tissue specimens and compared three different commercial library preparation kits, namely NEXTflex Rapid Directional qRNA-Seq Kit (Bioo Scientific), SENSE Total RNA-Seq Library Prep Kit (Lexogen) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB).

    Article Title: Transcriptome engineering with RNA-targeting Type VI-D CRISPR effectors
    Article Snippet: Paragraph title: RNA-seq library preparation and sequencing ... Stranded mRNA libraries were prepared using the NEBNext II Ultra Directional RNA Library Prep Kit from New England Biolabs (Cat# E7760S) and sequenced on an Illumina NextSeq500 with 42 nt paired end reads.

    Article Title: Transcriptome Analysis of Intrusively Growing Flax Fibers Isolated by Laser Microdissection
    Article Snippet: .. To produce normalized cDNA libraries, followed by sequencing using Illumina MiSeq with single-end 75 bp reads. cDNA libraries from total RNA of iFIB samples (up to 1 µg) were prepared with NEBNext Ultra II Directional RNA Library Prep Kit after selective depletion of ribosomal RNA using RiboMinus™ Plant Kit for RNA-Seq according to the manufacturer’s instructions. .. Sequence was performed on HiSeq2500 with 60 bp single-end reads.

    Generated:

    Article Title: Antiviral potential of human IFN-α subtypes against influenza A H3N2 infection in human lung explants reveals subtype-specific activities
    Article Snippet: .. The RNA sequencing library was generated from 500 ng total RNA using Dynabeads mRNA DIRECT Micro Purification Kit (Thermo Fisher Scientific, USA), for mRNA purification, followed by NEBNext Ultra II Directional RNA Library Prep Kit (New England BioLabs, USA), according to manufacturer’s protocols. .. The libraries were sequenced on Illumina NovaSeq 6000 using NovaSeq 6000 S1 Reagent Kit (200 and 300 cycles, paired end runs) with an average of 7.9 × 107 reads per sample.

    Article Title: Testing of library preparation methods for transcriptome sequencing of real life glioblastoma and brain tissue specimens: A comparative study with special focus on long non-coding RNAs
    Article Snippet: In the present study, we used GBM and non-tumor brain tissue specimens and compared three different commercial library preparation kits, namely NEXTflex Rapid Directional qRNA-Seq Kit (Bioo Scientific), SENSE Total RNA-Seq Library Prep Kit (Lexogen) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB). .. Libraries generated using SENSE kit were characterized by the most normal distribution of normalized average GC content, the least amount of over-represented sequences and the percentage of ribosomal RNA reads (0.3–1.5%) and highest numbers of uniquely mapped reads and reads aligning to coding regions.

    Polymerase Chain Reaction:

    Article Title: Testing of library preparation methods for transcriptome sequencing of real life glioblastoma and brain tissue specimens: A comparative study with special focus on long non-coding RNAs
    Article Snippet: .. Out of the three library preparation kits we tested, NEBNext Ultra II Directional RNA Library Prep for Illumina kit performed best with regard to relatively even transcript coverage, low rates of PCR duplicates and the highest number of hits for biotypes of interest in Ensembl database. ..

    Sample Prep:

    Article Title: Clonotypic heterogeneity in cutaneous T-cell lymphoma (mycosis fungoides) revealed by comprehensive whole-exome sequencing
    Article Snippet: Paragraph title: Sample preparation for whole-transcriptome sequencing ... Ribosomal RNA–depleted samples were used for complementary DNA (cDNA) synthesis, and the library was built using a NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (catalog number E7760; New England Biolabs).

    Article Title: Transcriptome Analysis of Intrusively Growing Flax Fibers Isolated by Laser Microdissection
    Article Snippet: For sequencing, total RNA from the APEX (1–3 µg) sample was processed by using TruSeq Sample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instruction. .. To produce normalized cDNA libraries, followed by sequencing using Illumina MiSeq with single-end 75 bp reads. cDNA libraries from total RNA of iFIB samples (up to 1 µg) were prepared with NEBNext Ultra II Directional RNA Library Prep Kit after selective depletion of ribosomal RNA using RiboMinus™ Plant Kit for RNA-Seq according to the manufacturer’s instructions.

    RNA Extraction:

    Article Title: Transcriptome Analysis of Intrusively Growing Flax Fibers Isolated by Laser Microdissection
    Article Snippet: Paragraph title: RNA extraction and sequencing ... To produce normalized cDNA libraries, followed by sequencing using Illumina MiSeq with single-end 75 bp reads. cDNA libraries from total RNA of iFIB samples (up to 1 µg) were prepared with NEBNext Ultra II Directional RNA Library Prep Kit after selective depletion of ribosomal RNA using RiboMinus™ Plant Kit for RNA-Seq according to the manufacturer’s instructions.

    Software:

    Article Title: Sensory neuron lineage mapping and manipulation in the Drosophila olfactory system
    Article Snippet: RNA-seq libraries were prepared from the mRNA using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). .. Sequencing data were demultiplexed using the bcl2fastq Conversion Software (version 2.20, Illumina).

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    New England Biolabs ultra ii directional rna library prep kit
    Loss of Pnt results in Naa-to-Nab transformations in diverse sensillar subtypes. a A sensillum can contain up to four OSNs through differentiation of Naa (cyan), Nab (magenta), Nba (green), Nbb (yellow) terminal daughter cells originating from a single SOP lineage. b Representative images of <t>RNA</t> FISH for Or67d (magenta) in at1 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or67d-expressing OSNs are duplicated (arrow). A schematic of the proposed Naa-to-Nab fate transformation is shown on the right (color scheme as in ( a )). Scale bar = 2 µm. The open circles in this and other schematics represent OSN precursors that have undergone apoptosis. c Representative images of RNA FISH for Or85a (magenta) and Or59b (green) in ab2 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or85a OSNs (Nab) are duplicated (arrow), while Or59b OSNs (Nba) are unaffected. d Representative images of RNA FISH for Or85b (magenta) and Or22a (green) in ab3 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or85b OSNs (Nab) are duplicated (arrow), while Or22a OSNs (Nba) are unaffected. e Representative images of RNA FISH for Or92a (magenta) and Or10a (cyan) in ab1 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or92a OSNs (Nab) are duplicated (arrow), while Or10a OSNs (Naa) are lost. f Top: theoretical ratios of OSN types in 2-, 3- and 4-neuron sensilla in control and pnt RNAi antennae, assuming Naa-to-Nab fate transformation (i.e. loss of Naa OSNs, and duplication of Nab OSNs). Bottom: experimentally determined OSN ratios in all sensilla in pnt RNAi antennae using as a proxy the normalized ratios of olfactory receptor <t>mRNA</t> expression from antennal transcriptomes (see Supplementary Fig. 6e ). In ab10, Or49a is reported to be coexpressed with Or85f 13 , but transcript levels for this gene were below the cut-off applied during the analysis of these RNA-seq datasets
    Ultra Ii Directional Rna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Loss of Pnt results in Naa-to-Nab transformations in diverse sensillar subtypes. a A sensillum can contain up to four OSNs through differentiation of Naa (cyan), Nab (magenta), Nba (green), Nbb (yellow) terminal daughter cells originating from a single SOP lineage. b Representative images of RNA FISH for Or67d (magenta) in at1 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or67d-expressing OSNs are duplicated (arrow). A schematic of the proposed Naa-to-Nab fate transformation is shown on the right (color scheme as in ( a )). Scale bar = 2 µm. The open circles in this and other schematics represent OSN precursors that have undergone apoptosis. c Representative images of RNA FISH for Or85a (magenta) and Or59b (green) in ab2 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or85a OSNs (Nab) are duplicated (arrow), while Or59b OSNs (Nba) are unaffected. d Representative images of RNA FISH for Or85b (magenta) and Or22a (green) in ab3 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or85b OSNs (Nab) are duplicated (arrow), while Or22a OSNs (Nba) are unaffected. e Representative images of RNA FISH for Or92a (magenta) and Or10a (cyan) in ab1 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or92a OSNs (Nab) are duplicated (arrow), while Or10a OSNs (Naa) are lost. f Top: theoretical ratios of OSN types in 2-, 3- and 4-neuron sensilla in control and pnt RNAi antennae, assuming Naa-to-Nab fate transformation (i.e. loss of Naa OSNs, and duplication of Nab OSNs). Bottom: experimentally determined OSN ratios in all sensilla in pnt RNAi antennae using as a proxy the normalized ratios of olfactory receptor mRNA expression from antennal transcriptomes (see Supplementary Fig. 6e ). In ab10, Or49a is reported to be coexpressed with Or85f 13 , but transcript levels for this gene were below the cut-off applied during the analysis of these RNA-seq datasets

    Journal: Nature Communications

    Article Title: Sensory neuron lineage mapping and manipulation in the Drosophila olfactory system

    doi: 10.1038/s41467-019-08345-4

    Figure Lengend Snippet: Loss of Pnt results in Naa-to-Nab transformations in diverse sensillar subtypes. a A sensillum can contain up to four OSNs through differentiation of Naa (cyan), Nab (magenta), Nba (green), Nbb (yellow) terminal daughter cells originating from a single SOP lineage. b Representative images of RNA FISH for Or67d (magenta) in at1 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or67d-expressing OSNs are duplicated (arrow). A schematic of the proposed Naa-to-Nab fate transformation is shown on the right (color scheme as in ( a )). Scale bar = 2 µm. The open circles in this and other schematics represent OSN precursors that have undergone apoptosis. c Representative images of RNA FISH for Or85a (magenta) and Or59b (green) in ab2 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or85a OSNs (Nab) are duplicated (arrow), while Or59b OSNs (Nba) are unaffected. d Representative images of RNA FISH for Or85b (magenta) and Or22a (green) in ab3 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or85b OSNs (Nab) are duplicated (arrow), while Or22a OSNs (Nba) are unaffected. e Representative images of RNA FISH for Or92a (magenta) and Or10a (cyan) in ab1 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or92a OSNs (Nab) are duplicated (arrow), while Or10a OSNs (Naa) are lost. f Top: theoretical ratios of OSN types in 2-, 3- and 4-neuron sensilla in control and pnt RNAi antennae, assuming Naa-to-Nab fate transformation (i.e. loss of Naa OSNs, and duplication of Nab OSNs). Bottom: experimentally determined OSN ratios in all sensilla in pnt RNAi antennae using as a proxy the normalized ratios of olfactory receptor mRNA expression from antennal transcriptomes (see Supplementary Fig. 6e ). In ab10, Or49a is reported to be coexpressed with Or85f 13 , but transcript levels for this gene were below the cut-off applied during the analysis of these RNA-seq datasets

    Article Snippet: RNA-seq libraries were prepared from the mRNA using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs).

    Techniques: Fluorescence In Situ Hybridization, Expressing, Transformation Assay, RNA Sequencing Assay

    An OSN lineage-specific driver. a Top row: developmental expression of the nonimmortalized GMR82D08-GAL4 (hereafter, at1 driver) using a myr:GFP reporter (green) in the antennal disc SOPs (region marked by α-Dac (blue)) during late larval/early pupal stages. Bottom row: the at1 driver is expressed in the daughter cells of these SOPs in the developing pupal antenna but progressively loses its expression from 20 h APF as OSNs differentiate (visualized with the neuronal marker α-Elav (magenta)). Scale bar = 20 µm in this and other panels. b Immortalization of the at1 driver reveals labeling of clusters of cells in the adult antenna by an rCD2:GFP reporter (green). RNA fluorescence in situ hybridization demonstrates that a single cell within each cluster (arrowheads in the inset images) expresses Or67d mRNA (magenta). c Representative example of a single sensillum in the adult antenna labeled by the immortalized at1 driver, viewed at three focal planes. There is a single Or67d mRNA-positive OSN (cell 1, arrowhead), flanked by four non-neuronal support cells (cells 2–5). d Sensilla cells labeled by the immortalized at1 driver lineage (α-GFP; green) also express Lush (magenta), an odorant binding protein unique to trichoid sensilla support cells 72

    Journal: Nature Communications

    Article Title: Sensory neuron lineage mapping and manipulation in the Drosophila olfactory system

    doi: 10.1038/s41467-019-08345-4

    Figure Lengend Snippet: An OSN lineage-specific driver. a Top row: developmental expression of the nonimmortalized GMR82D08-GAL4 (hereafter, at1 driver) using a myr:GFP reporter (green) in the antennal disc SOPs (region marked by α-Dac (blue)) during late larval/early pupal stages. Bottom row: the at1 driver is expressed in the daughter cells of these SOPs in the developing pupal antenna but progressively loses its expression from 20 h APF as OSNs differentiate (visualized with the neuronal marker α-Elav (magenta)). Scale bar = 20 µm in this and other panels. b Immortalization of the at1 driver reveals labeling of clusters of cells in the adult antenna by an rCD2:GFP reporter (green). RNA fluorescence in situ hybridization demonstrates that a single cell within each cluster (arrowheads in the inset images) expresses Or67d mRNA (magenta). c Representative example of a single sensillum in the adult antenna labeled by the immortalized at1 driver, viewed at three focal planes. There is a single Or67d mRNA-positive OSN (cell 1, arrowhead), flanked by four non-neuronal support cells (cells 2–5). d Sensilla cells labeled by the immortalized at1 driver lineage (α-GFP; green) also express Lush (magenta), an odorant binding protein unique to trichoid sensilla support cells 72

    Article Snippet: RNA-seq libraries were prepared from the mRNA using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs).

    Techniques: Expressing, Marker, Labeling, Fluorescence, In Situ Hybridization, Binding Assay

    An OSN lineage-specific driver. a Top row: developmental expression of the nonimmortalized GMR82D08-GAL4 (hereafter, at1 driver) using a myr:GFP reporter (green) in the antennal disc SOPs (region marked by α-Dac (blue)) during late larval/early pupal stages. Bottom row: the at1 driver is expressed in the daughter cells of these SOPs in the developing pupal antenna but progressively loses its expression from 20 h APF as OSNs differentiate (visualized with the neuronal marker α-Elav (magenta)). Scale bar = 20 µm in this and other panels. b Immortalization of the at1 driver reveals labeling of clusters of cells in the adult antenna by an rCD2:GFP reporter (green). RNA fluorescence in situ hybridization demonstrates that a single cell within each cluster (arrowheads in the inset images) expresses Or67d mRNA (magenta). c Representative example of a single sensillum in the adult antenna labeled by the immortalized at1 driver, viewed at three focal planes. There is a single Or67d mRNA-positive OSN (cell 1, arrowhead), flanked by four non-neuronal support cells (cells 2–5). d

    Journal: Nature Communications

    Article Title: Sensory neuron lineage mapping and manipulation in the Drosophila olfactory system

    doi: 10.1038/s41467-019-08345-4

    Figure Lengend Snippet: An OSN lineage-specific driver. a Top row: developmental expression of the nonimmortalized GMR82D08-GAL4 (hereafter, at1 driver) using a myr:GFP reporter (green) in the antennal disc SOPs (region marked by α-Dac (blue)) during late larval/early pupal stages. Bottom row: the at1 driver is expressed in the daughter cells of these SOPs in the developing pupal antenna but progressively loses its expression from 20 h APF as OSNs differentiate (visualized with the neuronal marker α-Elav (magenta)). Scale bar = 20 µm in this and other panels. b Immortalization of the at1 driver reveals labeling of clusters of cells in the adult antenna by an rCD2:GFP reporter (green). RNA fluorescence in situ hybridization demonstrates that a single cell within each cluster (arrowheads in the inset images) expresses Or67d mRNA (magenta). c Representative example of a single sensillum in the adult antenna labeled by the immortalized at1 driver, viewed at three focal planes. There is a single Or67d mRNA-positive OSN (cell 1, arrowhead), flanked by four non-neuronal support cells (cells 2–5). d

    Article Snippet: RNA-seq libraries were prepared from the mRNA using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs).

    Techniques: Expressing, Marker, Labeling, Fluorescence, In Situ Hybridization

    Results from comparison of three library preparation kits using the Ensembl automatic annotation system. The number of hits for each transcript biotype from Ensembl database calculated for each group of libraries generated using the same library preparation kit is on y-axis. Only the most populous biotypes relevant to our current study were included. While the first biotype comprises the majority of the group of protein coding RNA, the rest (lincRNA, antisense and sense intronic RNA) is part of another large group of long non-coding RNA. After comparing all three kits, the highest number of hits for each biotype of interest was observed for NEBNext (NB) libraries; NEXTflex (NF) libraries came second and SENSE (LX) libraries third. These results hint at a lesser variability of library fragments in LX libraries.

    Journal: PLoS ONE

    Article Title: Testing of library preparation methods for transcriptome sequencing of real life glioblastoma and brain tissue specimens: A comparative study with special focus on long non-coding RNAs

    doi: 10.1371/journal.pone.0211978

    Figure Lengend Snippet: Results from comparison of three library preparation kits using the Ensembl automatic annotation system. The number of hits for each transcript biotype from Ensembl database calculated for each group of libraries generated using the same library preparation kit is on y-axis. Only the most populous biotypes relevant to our current study were included. While the first biotype comprises the majority of the group of protein coding RNA, the rest (lincRNA, antisense and sense intronic RNA) is part of another large group of long non-coding RNA. After comparing all three kits, the highest number of hits for each biotype of interest was observed for NEBNext (NB) libraries; NEXTflex (NF) libraries came second and SENSE (LX) libraries third. These results hint at a lesser variability of library fragments in LX libraries.

    Article Snippet: In the present study, we used GBM and non-tumor brain tissue specimens and compared three different commercial library preparation kits, namely NEXTflex Rapid Directional qRNA-Seq Kit (Bioo Scientific), SENSE Total RNA-Seq Library Prep Kit (Lexogen) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB).

    Techniques: Generated

    ( A ) Overrepresentation analysis of up- and down-regulated genes within functional gene classes defined by MapMan bins (MapMan, v3.6.0RC1, available online at http://mapman.gabipd.org/web/guest/mapman [accessed on 29 June 2018]) in iFIB sample as compared to APEX (iFIB/APEX). The data were subjected to a Wilcoxon test; resulting p-values were adjusted according to Benjamini and Hochberg in PageMan ( http://mapman.gabipd.org/pageman [accessed on 20 July 2018]), and the results are displayed in false color. Functional gene classes colored in red are significantly up-regulated, whereas ones colored in green are significantly down-regulated. PS, photosynthesis; TCA, tricarboxylic acid cycle; AA, amino acid; CHO, carbohydrate. ( B ) Verification of RNA-Seq expression through qRT-PCR, error bar shows the standard error of the mean.

    Journal: Scientific Reports

    Article Title: Transcriptome Analysis of Intrusively Growing Flax Fibers Isolated by Laser Microdissection

    doi: 10.1038/s41598-018-32869-2

    Figure Lengend Snippet: ( A ) Overrepresentation analysis of up- and down-regulated genes within functional gene classes defined by MapMan bins (MapMan, v3.6.0RC1, available online at http://mapman.gabipd.org/web/guest/mapman [accessed on 29 June 2018]) in iFIB sample as compared to APEX (iFIB/APEX). The data were subjected to a Wilcoxon test; resulting p-values were adjusted according to Benjamini and Hochberg in PageMan ( http://mapman.gabipd.org/pageman [accessed on 20 July 2018]), and the results are displayed in false color. Functional gene classes colored in red are significantly up-regulated, whereas ones colored in green are significantly down-regulated. PS, photosynthesis; TCA, tricarboxylic acid cycle; AA, amino acid; CHO, carbohydrate. ( B ) Verification of RNA-Seq expression through qRT-PCR, error bar shows the standard error of the mean.

    Article Snippet: To produce normalized cDNA libraries, followed by sequencing using Illumina MiSeq with single-end 75 bp reads. cDNA libraries from total RNA of iFIB samples (up to 1 µg) were prepared with NEBNext Ultra II Directional RNA Library Prep Kit after selective depletion of ribosomal RNA using RiboMinus™ Plant Kit for RNA-Seq according to the manufacturer’s instructions.

    Techniques: Functional Assay, RNA Sequencing Assay, Expressing, Quantitative RT-PCR

    Scheme of plant material collection to obtain APEX, iFIB, FIB samples for subsequent RNA-Seq analysis. Bundles of the intrusively growing fibers (sample iFIB) were isolated by laser microdissection from the longitudinal cryosections of 3rd cm from the stem apex. Sample APEX was collected as 2 mm of the uppermost stem together with leaf primordia. The described earlier 12 sample FIB (fibers isolated during tertiary cell wall deposition) was collected from the stem portion below the snap point (SP).

    Journal: Scientific Reports

    Article Title: Transcriptome Analysis of Intrusively Growing Flax Fibers Isolated by Laser Microdissection

    doi: 10.1038/s41598-018-32869-2

    Figure Lengend Snippet: Scheme of plant material collection to obtain APEX, iFIB, FIB samples for subsequent RNA-Seq analysis. Bundles of the intrusively growing fibers (sample iFIB) were isolated by laser microdissection from the longitudinal cryosections of 3rd cm from the stem apex. Sample APEX was collected as 2 mm of the uppermost stem together with leaf primordia. The described earlier 12 sample FIB (fibers isolated during tertiary cell wall deposition) was collected from the stem portion below the snap point (SP).

    Article Snippet: To produce normalized cDNA libraries, followed by sequencing using Illumina MiSeq with single-end 75 bp reads. cDNA libraries from total RNA of iFIB samples (up to 1 µg) were prepared with NEBNext Ultra II Directional RNA Library Prep Kit after selective depletion of ribosomal RNA using RiboMinus™ Plant Kit for RNA-Seq according to the manufacturer’s instructions.

    Techniques: RNA Sequencing Assay, Isolation, Laser Capture Microdissection