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    New England Biolabs nebnext ultra rna library prep kit for illumina
    Optimization of SHERRY and comparison with <t>NEBNext.</t> ( A ) Gene number detected by SHERRY under various experimental conditions. Each condition consisted of three replicates of 10 ng HEK293T total <t>RNA.</t> ( B ) Comparison of sequencing indicators between SHERRY and NEBNext with 10 ng and 200 ng HEK293T total RNA input. Each condition consisted of three replicates and down-sampled to 2 million total reads.
    Nebnext Ultra Rna Library Prep Kit For Illumina, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1770 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nebnext ultra rna library prep kit for illumina/product/New England Biolabs
    Average 99 stars, based on 1770 article reviews
    Price from $9.99 to $1999.99
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    99
    New England Biolabs ultra directional rna library prep kit
    Internode zones have unique <t>RNAseq</t> profiles and contain stem- and zone-specific genes. a Principal component analysis of FPKM values obtained from <t>RNA</t> sequencing of the MsZ, CEZ, TZ, and MZ of internode 5. b Venn diagram of genes up-regulated greater than a log 2 fold change of one against the background levels in the whole plant (WP) transcriptome. c Table showing the number of genes expressed > 1 FPKM, number of genes up-regulated against the WP transcriptome with > 1 log 2 fold, number of identified ‘stem-specific’ genes with > 2.5 log 2 fold change against the WP background and > 80 FPKM in any internode zone, and the number of ‘zone-specific’ genes with, in addition to being ‘stem-specific’, > 2 log 2 fold change against all other internodal zones (with some exceptions, see Additional file 2 )
    Ultra Directional Rna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 6019 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ultra directional rna library prep kit/product/New England Biolabs
    Average 99 stars, based on 6019 article reviews
    Price from $9.99 to $1999.99
    ultra directional rna library prep kit - by Bioz Stars, 2020-09
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    99
    New England Biolabs nebnext ultra ii directional rna library prep kit for illumina
    Transcriptomic analysis of the sre1 Δ and wild-type strains in response to alkaline pH. WT and sre1 Δ cells were incubated in YPD medium pH 4 or pH 8 for 90 min. This experiment was conducted with six biological replicates for each strain and condition. Total <t>RNA</t> was extracted, mRNA was isolated, and libraries were prepared and finally sequenced using an <t>Illumina</t> NextSeq 500 sequencer. GO-term analysis was performed using FungiDB. (A) The majority of the known genes in C. neoformans ergosterol biosynthesis were significantly differentially expressed in the sre1 Δ versus wild-type transcriptome at pH 8. ERG genes that were significantly differentially expressed have an adjusted P value of
    Nebnext Ultra Ii Directional Rna Library Prep Kit For Illumina, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 539 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 539 article reviews
    Price from $9.99 to $1999.99
    nebnext ultra ii directional rna library prep kit for illumina - by Bioz Stars, 2020-09
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    99
    New England Biolabs nebnext ultra ii rna library prep kit for illumina
    Quality assessment of TRACE-seq. ( a ) Gene expression measured by two technical replicates of TRACE-seq with 20 ng total <t>RNA</t> as input are shown as scatter plots. Pearson's product-moment correlations are displayed in the upper left corner. ( b ) Gene expression measured by two technical replicates of TRACE-seq with 2 ng total RNA as input are shown as scatter plots. Pearson's product-moment correlations are displayed in the upper left corner. ( c ) Scatterplots showing gene expression values for TRACE-seq and <t>NEBNext</t> Ultra II RNA kit with 10 ng mRNA as input (left), and for TRACE-seq with 10 ng mRNA as input and Smart-seq2 with 20 ng total RNA as input (right). All expressed genes (FPKM > 0.5) are included. Pearson's product-moment correlation is displayed in the upper left corner. ( d ) Distribution of the insert size in NEBNext Ultra II RNA library, Smart-seq2 libraries and TRACE-seq libraries with different amount of RNA as input, respectively. ( e ) Median coefficient of variation of gene coverage over the 1000 most highly expressed transcripts in NEBNext Ultra II RNA library, Smart-seq2 libraries and TRACE-seq libraries with different amount of RNA as input, respectively. ( f ) Comparison of read coverage over gene body for NEBNext Ultra II RNA kit, Smart-seq2 and TRACE-seq with different amount of RNA as input. Transcripts were grouped according to annotated lengths and analyzed separately, with the transcript length ranges indicated (top right). The read coverage over gene body is displayed along with gene body percentile from 5’ to 3’ end. ( g ) Library complexity for each library shown by plotting the number of uniquely occurring read-pairs with respect to total number of sampled read-pairs. ( h ) Assessment of RNA Integrity number (RIN). RNA of high RIN score (9.5) was used as input, which allows us to rule out the possibility that the 3’ end bias of gene body coverage is due to RNA degradation. ( i ) Nucleotide versus cycle (NVC) plots showing percentage of observed bases at each position of the first 30 bases of each sequencing read from TRACE-seq library with 10 ng mRNA and 20 ng total RNA as input. ( j ) WebLogo plot of sequence conservations of the first 10 bases of all sequencing reads from TRACE-seq library with 10 ng mRNA as input. The overall height of the stack indicates the sequence conservation at that position (measured in bits), while the height of symbols within the stack indicates the relative frequency of each nucleic acid at that position.
    Nebnext Ultra Ii Rna Library Prep Kit For Illumina, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 288 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nebnext ultra ii rna library prep kit for illumina/product/New England Biolabs
    Average 99 stars, based on 288 article reviews
    Price from $9.99 to $1999.99
    nebnext ultra ii rna library prep kit for illumina - by Bioz Stars, 2020-09
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    Image Search Results


    Optimization of SHERRY and comparison with NEBNext. ( A ) Gene number detected by SHERRY under various experimental conditions. Each condition consisted of three replicates of 10 ng HEK293T total RNA. ( B ) Comparison of sequencing indicators between SHERRY and NEBNext with 10 ng and 200 ng HEK293T total RNA input. Each condition consisted of three replicates and down-sampled to 2 million total reads.

    Journal: bioRxiv

    Article Title: RNA Sequencing by Direct Tagmentation of RNA/DNA Hybrids

    doi: 10.1101/843474

    Figure Lengend Snippet: Optimization of SHERRY and comparison with NEBNext. ( A ) Gene number detected by SHERRY under various experimental conditions. Each condition consisted of three replicates of 10 ng HEK293T total RNA. ( B ) Comparison of sequencing indicators between SHERRY and NEBNext with 10 ng and 200 ng HEK293T total RNA input. Each condition consisted of three replicates and down-sampled to 2 million total reads.

    Article Snippet: Then we plotted a heatmap of the distance matrix ( ) between different cell types and library preparation methods.

    Techniques: Sequencing

    Coverage evenness optimization of SHERRY (10 ng HEK293T total RNA input). ( A ) Normalized transcript coverage of standard SHERRY, SHERRY using TSO-RT method and NEBNext kit. ( B ) The coverage of GAPDH transcript calculated from SHERRY and TSO-RT SHERRY. ( C ) Comparison of sequencing indicators between SHERRY (n=3) and TSO-RT SHERRY (n=2).

    Journal: bioRxiv

    Article Title: RNA Sequencing by Direct Tagmentation of RNA/DNA Hybrids

    doi: 10.1101/843474

    Figure Lengend Snippet: Coverage evenness optimization of SHERRY (10 ng HEK293T total RNA input). ( A ) Normalized transcript coverage of standard SHERRY, SHERRY using TSO-RT method and NEBNext kit. ( B ) The coverage of GAPDH transcript calculated from SHERRY and TSO-RT SHERRY. ( C ) Comparison of sequencing indicators between SHERRY (n=3) and TSO-RT SHERRY (n=2).

    Article Snippet: Then we plotted a heatmap of the distance matrix ( ) between different cell types and library preparation methods.

    Techniques: Sequencing

    Functional comparison between SHERRY and NEBNext. ( A ) Correlation of normalized gene counts among duplicates of SHERRY, which start from 200 ng HEK293T total R NA input. ( B ) Correlation of normalized genes counts (average of three replicates) between SHERRY and NEBNext within the two cell types. The input was 200 ng total RNA. ( C ) Differentially expressed genes of HeLa and HEK293T detected by SHERRY and NEBNext kit (200 ng input) are plotted into Venn Diagram. Colored area represents genes identified by both methods. Gene numbers are listed on corresponding part. ( D ) Heatmap of differentially expressed genes detected by SHERRY while missed by NEBNext kit. The Color bar indicates Z-score. ( E ) Heatmap of differentially expressed genes detected by NEBNext kit while missed by SHERRY. The Color bar indicates Z-score.

    Journal: bioRxiv

    Article Title: RNA Sequencing by Direct Tagmentation of RNA/DNA Hybrids

    doi: 10.1101/843474

    Figure Lengend Snippet: Functional comparison between SHERRY and NEBNext. ( A ) Correlation of normalized gene counts among duplicates of SHERRY, which start from 200 ng HEK293T total R NA input. ( B ) Correlation of normalized genes counts (average of three replicates) between SHERRY and NEBNext within the two cell types. The input was 200 ng total RNA. ( C ) Differentially expressed genes of HeLa and HEK293T detected by SHERRY and NEBNext kit (200 ng input) are plotted into Venn Diagram. Colored area represents genes identified by both methods. Gene numbers are listed on corresponding part. ( D ) Heatmap of differentially expressed genes detected by SHERRY while missed by NEBNext kit. The Color bar indicates Z-score. ( E ) Heatmap of differentially expressed genes detected by NEBNext kit while missed by SHERRY. The Color bar indicates Z-score.

    Article Snippet: Then we plotted a heatmap of the distance matrix ( ) between different cell types and library preparation methods.

    Techniques: Functional Assay

    Performance of SHERRY with large RNA input. ( A ) Coefficient of variation (CV) across three replicates was plotted against the mean value of each gene’s FPKM (Fragments Per Kilobase of transcript per Million mapped reads). All experiments used HEK293T total RNA as input. ( B ) Genes detected by SHERRY in three replicates of 200 ng HEK293T or HeLa total RNA are plotted in Venn Diagrams. Numbers of common genes are indicated. ( C ) Common genes detected by SHERRY and NEBNext in the three replicates of 200 ng HEK293T or HeLa total RNA. ( D ) Distance heatmap of samples prepared by SHERRY or NEBNext for three replicates using 200 ng HEK293T or HeLa total RNA. The color bar indicates the Euclidian distance. ( E ) Correlation of gene expression fold-change identified by SHERRY and NEBNext. Involved genes are differentially expressed genes between HEK293T and HeLa detected by both methods.

    Journal: bioRxiv

    Article Title: RNA Sequencing by Direct Tagmentation of RNA/DNA Hybrids

    doi: 10.1101/843474

    Figure Lengend Snippet: Performance of SHERRY with large RNA input. ( A ) Coefficient of variation (CV) across three replicates was plotted against the mean value of each gene’s FPKM (Fragments Per Kilobase of transcript per Million mapped reads). All experiments used HEK293T total RNA as input. ( B ) Genes detected by SHERRY in three replicates of 200 ng HEK293T or HeLa total RNA are plotted in Venn Diagrams. Numbers of common genes are indicated. ( C ) Common genes detected by SHERRY and NEBNext in the three replicates of 200 ng HEK293T or HeLa total RNA. ( D ) Distance heatmap of samples prepared by SHERRY or NEBNext for three replicates using 200 ng HEK293T or HeLa total RNA. The color bar indicates the Euclidian distance. ( E ) Correlation of gene expression fold-change identified by SHERRY and NEBNext. Involved genes are differentially expressed genes between HEK293T and HeLa detected by both methods.

    Article Snippet: Then we plotted a heatmap of the distance matrix ( ) between different cell types and library preparation methods.

    Techniques: Expressing

    mRNA-seq reveals relative contributions of current environment and ancestral environment in shaping gene expression variation. (A) Schematic of experimental set-up for collection of RNA-seq samples. (B) Principal component analysis (PCA) of six conditions including all 8649 reliably detected genes; 97% of variance explained by whether worms were fed or starved at collection (PC1), and 1.3% of variance explained by whether ancestors experienced control, short-term dauer, or long-term dauer conditions (PC2). Mean CPM of biological replicates were used for each condition for PCA. Number of biological replicates: control starved (12), ST dauer starved (9), LT dauer starved (6), control fed (4), ST dauer fed (4), and LT dauer fed (3).

    Journal: Genetics

    Article Title: Transgenerational Effects of Extended Dauer Diapause on Starvation Survival and Gene Expression Plasticity in Caenorhabditis elegans

    doi: 10.1534/genetics.118.301250

    Figure Lengend Snippet: mRNA-seq reveals relative contributions of current environment and ancestral environment in shaping gene expression variation. (A) Schematic of experimental set-up for collection of RNA-seq samples. (B) Principal component analysis (PCA) of six conditions including all 8649 reliably detected genes; 97% of variance explained by whether worms were fed or starved at collection (PC1), and 1.3% of variance explained by whether ancestors experienced control, short-term dauer, or long-term dauer conditions (PC2). Mean CPM of biological replicates were used for each condition for PCA. Number of biological replicates: control starved (12), ST dauer starved (9), LT dauer starved (6), control fed (4), ST dauer fed (4), and LT dauer fed (3).

    Article Snippet: RNA was isolated using TRIzol (Invitrogen) using the manufacturer’s protocol with minor modifications; 1 ml of TRIzol was used per sample along with 100 μl of acid-washed sand. mRNA-seq libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina (E7530) in two batches, utilizing either 500 or 100 ng of starting RNA per library and 12 or 15 PCR cycles, respectively.

    Techniques: Expressing, RNA Sequencing Assay

    RBM4 and Nova1 exert differential effects on splicing profiles of the Nova1 gene. ( a ) The diagram presents the sequence of human and mouse Nova1 exon 4 and flanking introns. The responsive elements of Nova1 (underlined black characters) and RBM4 (underlined gray characters) are underlined. HCT-8 cells were transfected with overexpressing or targeting vectors, followed by total RNA and lysate extraction. Splicing profiles of Nova1 transcripts were analyzed with specific primer sets. Immunoblot assays of indicated proteins were analyzed with specific antibodies. The bar graph shows relative levels of Nova1 − 4 in three independent experiments. ( b ) Nova1 minigenes were cotransfected with expressing vectors into HCT-8 cells. Spliced transcripts of Nova1 minigenes were analyzed using SV40 oligo and a specific Nova1 primer as described in the previous panel. The bar graph shows the relative level of Nova1 − 4 in three independent experiments using TotalLab Quant Software. The quantitative results are shown as the mean ± STD ( p

    Journal: Scientific Reports

    Article Title: The impact of the RBM4-initiated splicing cascade on modulating the carcinogenic signature of colorectal cancer cells

    doi: 10.1038/srep44204

    Figure Lengend Snippet: RBM4 and Nova1 exert differential effects on splicing profiles of the Nova1 gene. ( a ) The diagram presents the sequence of human and mouse Nova1 exon 4 and flanking introns. The responsive elements of Nova1 (underlined black characters) and RBM4 (underlined gray characters) are underlined. HCT-8 cells were transfected with overexpressing or targeting vectors, followed by total RNA and lysate extraction. Splicing profiles of Nova1 transcripts were analyzed with specific primer sets. Immunoblot assays of indicated proteins were analyzed with specific antibodies. The bar graph shows relative levels of Nova1 − 4 in three independent experiments. ( b ) Nova1 minigenes were cotransfected with expressing vectors into HCT-8 cells. Spliced transcripts of Nova1 minigenes were analyzed using SV40 oligo and a specific Nova1 primer as described in the previous panel. The bar graph shows the relative level of Nova1 − 4 in three independent experiments using TotalLab Quant Software. The quantitative results are shown as the mean ± STD ( p

    Article Snippet: Poly(A) messenger (m)RNAs were enriched from 8 μg total RNAs with a high integrity number (RIN > 8.0) using oligo (dT)-based affinity matrices, followed by library construction using the NEB Next Ultra RNA Library Prep Kit from Illumina (NEB, Ipswich, MA, USA) according to the manufacturer’s instructions.

    Techniques: Sequencing, Transfection, Expressing, Software

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

    Journal: Biotechnology for Biofuels

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

    doi: 10.1186/s13068-016-0457-6

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

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

    Techniques: RNA Sequencing Assay

    Schematic overview of Hi-GRIL-seq. Induction of T4 RNA ligase expression from the P tac promoter with IPTG leads to the expression of the enzyme and the formation of chimeras between base paired endogenous sRNAs and their targets. Following isolation of total RNA and rRNA depletion, a cDNA library for Illumina sequencing is constructed and sequenced. RNA interactions between sRNAs and their targets are identified by a BLAST-based analysis pipeline. Global chimeras are visualized in a two-dimensional dot plot, in which the location of the dot represents the genomic coordinate of the participating RNAs. To examine the targets of a particular RNA, the coverage of its targets can be visualized. To further zoom in on a particular interaction between the two RNAs, the exact location of ligation junctions in the chimeras are mapped and visualized.

    Journal: Molecular microbiology

    Article Title: Probing the sRNA regulatory landscape of P. aeruginosa: post-transcriptional control of determinants of pathogenicity and antibiotic susceptibility

    doi: 10.1111/mmi.13857

    Figure Lengend Snippet: Schematic overview of Hi-GRIL-seq. Induction of T4 RNA ligase expression from the P tac promoter with IPTG leads to the expression of the enzyme and the formation of chimeras between base paired endogenous sRNAs and their targets. Following isolation of total RNA and rRNA depletion, a cDNA library for Illumina sequencing is constructed and sequenced. RNA interactions between sRNAs and their targets are identified by a BLAST-based analysis pipeline. Global chimeras are visualized in a two-dimensional dot plot, in which the location of the dot represents the genomic coordinate of the participating RNAs. To examine the targets of a particular RNA, the coverage of its targets can be visualized. To further zoom in on a particular interaction between the two RNAs, the exact location of ligation junctions in the chimeras are mapped and visualized.

    Article Snippet: After rRNA depletion or sRNA enrichment, cDNA libraries were prepared with the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs).

    Techniques: Expressing, Isolation, cDNA Library Assay, Sequencing, Construct, Ligation

    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

    Transcriptomic analysis of the sre1 Δ and wild-type strains in response to alkaline pH. WT and sre1 Δ cells were incubated in YPD medium pH 4 or pH 8 for 90 min. This experiment was conducted with six biological replicates for each strain and condition. Total RNA was extracted, mRNA was isolated, and libraries were prepared and finally sequenced using an Illumina NextSeq 500 sequencer. GO-term analysis was performed using FungiDB. (A) The majority of the known genes in C. neoformans ergosterol biosynthesis were significantly differentially expressed in the sre1 Δ versus wild-type transcriptome at pH 8. ERG genes that were significantly differentially expressed have an adjusted P value of

    Journal: mBio

    Article Title: Sterol-Response Pathways Mediate Alkaline Survival in Diverse Fungi

    doi: 10.1128/mBio.00719-20

    Figure Lengend Snippet: Transcriptomic analysis of the sre1 Δ and wild-type strains in response to alkaline pH. WT and sre1 Δ cells were incubated in YPD medium pH 4 or pH 8 for 90 min. This experiment was conducted with six biological replicates for each strain and condition. Total RNA was extracted, mRNA was isolated, and libraries were prepared and finally sequenced using an Illumina NextSeq 500 sequencer. GO-term analysis was performed using FungiDB. (A) The majority of the known genes in C. neoformans ergosterol biosynthesis were significantly differentially expressed in the sre1 Δ versus wild-type transcriptome at pH 8. ERG genes that were significantly differentially expressed have an adjusted P value of

    Article Snippet: The NEBNext poly(A) mRNA magnetic isolation module was used to enrich for mRNA, and the NEBNext Ultra II directional RNA library prep kit for Illumina was used to prepare libraries (New England Biolabs, Ipswich, MA).

    Techniques: Incubation, Isolation

    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

    Quality assessment of TRACE-seq. ( a ) Gene expression measured by two technical replicates of TRACE-seq with 20 ng total RNA as input are shown as scatter plots. Pearson's product-moment correlations are displayed in the upper left corner. ( b ) Gene expression measured by two technical replicates of TRACE-seq with 2 ng total RNA as input are shown as scatter plots. Pearson's product-moment correlations are displayed in the upper left corner. ( c ) Scatterplots showing gene expression values for TRACE-seq and NEBNext Ultra II RNA kit with 10 ng mRNA as input (left), and for TRACE-seq with 10 ng mRNA as input and Smart-seq2 with 20 ng total RNA as input (right). All expressed genes (FPKM > 0.5) are included. Pearson's product-moment correlation is displayed in the upper left corner. ( d ) Distribution of the insert size in NEBNext Ultra II RNA library, Smart-seq2 libraries and TRACE-seq libraries with different amount of RNA as input, respectively. ( e ) Median coefficient of variation of gene coverage over the 1000 most highly expressed transcripts in NEBNext Ultra II RNA library, Smart-seq2 libraries and TRACE-seq libraries with different amount of RNA as input, respectively. ( f ) Comparison of read coverage over gene body for NEBNext Ultra II RNA kit, Smart-seq2 and TRACE-seq with different amount of RNA as input. Transcripts were grouped according to annotated lengths and analyzed separately, with the transcript length ranges indicated (top right). The read coverage over gene body is displayed along with gene body percentile from 5’ to 3’ end. ( g ) Library complexity for each library shown by plotting the number of uniquely occurring read-pairs with respect to total number of sampled read-pairs. ( h ) Assessment of RNA Integrity number (RIN). RNA of high RIN score (9.5) was used as input, which allows us to rule out the possibility that the 3’ end bias of gene body coverage is due to RNA degradation. ( i ) Nucleotide versus cycle (NVC) plots showing percentage of observed bases at each position of the first 30 bases of each sequencing read from TRACE-seq library with 10 ng mRNA and 20 ng total RNA as input. ( j ) WebLogo plot of sequence conservations of the first 10 bases of all sequencing reads from TRACE-seq library with 10 ng mRNA as input. The overall height of the stack indicates the sequence conservation at that position (measured in bits), while the height of symbols within the stack indicates the relative frequency of each nucleic acid at that position.

    Journal: eLife

    Article Title: Transposase-assisted tagmentation of RNA/DNA hybrid duplexes

    doi: 10.7554/eLife.54919

    Figure Lengend Snippet: Quality assessment of TRACE-seq. ( a ) Gene expression measured by two technical replicates of TRACE-seq with 20 ng total RNA as input are shown as scatter plots. Pearson's product-moment correlations are displayed in the upper left corner. ( b ) Gene expression measured by two technical replicates of TRACE-seq with 2 ng total RNA as input are shown as scatter plots. Pearson's product-moment correlations are displayed in the upper left corner. ( c ) Scatterplots showing gene expression values for TRACE-seq and NEBNext Ultra II RNA kit with 10 ng mRNA as input (left), and for TRACE-seq with 10 ng mRNA as input and Smart-seq2 with 20 ng total RNA as input (right). All expressed genes (FPKM > 0.5) are included. Pearson's product-moment correlation is displayed in the upper left corner. ( d ) Distribution of the insert size in NEBNext Ultra II RNA library, Smart-seq2 libraries and TRACE-seq libraries with different amount of RNA as input, respectively. ( e ) Median coefficient of variation of gene coverage over the 1000 most highly expressed transcripts in NEBNext Ultra II RNA library, Smart-seq2 libraries and TRACE-seq libraries with different amount of RNA as input, respectively. ( f ) Comparison of read coverage over gene body for NEBNext Ultra II RNA kit, Smart-seq2 and TRACE-seq with different amount of RNA as input. Transcripts were grouped according to annotated lengths and analyzed separately, with the transcript length ranges indicated (top right). The read coverage over gene body is displayed along with gene body percentile from 5’ to 3’ end. ( g ) Library complexity for each library shown by plotting the number of uniquely occurring read-pairs with respect to total number of sampled read-pairs. ( h ) Assessment of RNA Integrity number (RIN). RNA of high RIN score (9.5) was used as input, which allows us to rule out the possibility that the 3’ end bias of gene body coverage is due to RNA degradation. ( i ) Nucleotide versus cycle (NVC) plots showing percentage of observed bases at each position of the first 30 bases of each sequencing read from TRACE-seq library with 10 ng mRNA and 20 ng total RNA as input. ( j ) WebLogo plot of sequence conservations of the first 10 bases of all sequencing reads from TRACE-seq library with 10 ng mRNA as input. The overall height of the stack indicates the sequence conservation at that position (measured in bits), while the height of symbols within the stack indicates the relative frequency of each nucleic acid at that position.

    Article Snippet: NEBNext and Smart-seq2 library preparationNEBNext Ultra II RNA libraries were constructed using NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB, #E7770S) according to the manufacturer’s instructions.

    Techniques: Expressing, Sequencing

    Performance of TRACE-seq in differential expression analysis. ( a ) Volcano plot showing differential expressed genes between undifferentiated and differentiated mESCs detected by NEBNext Ultra II RNA kit and TRACE-seq. Significantly up-regulated and down-regulated expressed genes (padj

    Journal: eLife

    Article Title: Transposase-assisted tagmentation of RNA/DNA hybrid duplexes

    doi: 10.7554/eLife.54919

    Figure Lengend Snippet: Performance of TRACE-seq in differential expression analysis. ( a ) Volcano plot showing differential expressed genes between undifferentiated and differentiated mESCs detected by NEBNext Ultra II RNA kit and TRACE-seq. Significantly up-regulated and down-regulated expressed genes (padj

    Article Snippet: NEBNext and Smart-seq2 library preparationNEBNext Ultra II RNA libraries were constructed using NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB, #E7770S) according to the manufacturer’s instructions.

    Techniques: Expressing

    Loss of DNA MMR activity reduces the innate antiviral transcriptional response against influenza A virus. (a) NanoLuc reporter expression and (b) relative cell viability in H441 cells that have been treated with PBS or H2O2 (for 30 min). Data shown as mean ± SD, n=4 independent samples. (c) Fold change of Mx1 RNA levels in H441 cells following treatment with PBS or IFN-alpha +/− H2O2 treatment (for 30 min). Data shown as mean ± SD, n=4 independent samples. (d) Western blot for Mx1 in H441 cells following the specified treatments. Tubulin = loading control. (e) NanoLuc reporter expression and (f) relative cell viability in H441 cells following the specified treatments. Data shown as mean ± SD, n=4 independent samples. (g) Median fluorescent intensity of the ISRE-GFP reporter in 293T cells following the specified treatments. Data shown as mean ± SD, n=3 independent samples. (h) Model depicting the role of DNA MMR in preserving antiviral gene expression. (i) RNAseq data showing fold change of mRNA levels in H441 cells comparing PR8-infected cells transfected with non-targeting siRNA (black) or MSH2+MSH6 siRNA (blue) to mock-infected cells. Inset is a magnified view of all genes induced > 5-fold in PR8-infected cells treated with non-targeting siRNA. (j) Chart grouping all of the genes induced > 5-fold in PR8-infected cells based on the effect MMR knockdown has on their mRNA levels. (k) Heat map displaying the effect of MMR knockdown on ISG and antiviral genes from the group of genes displayed in j. (l-o) Fold induction of (l) IFI44L and (n) IFIT1 RNA levels after viral infection as well as the difference in infection-induced (m) IFI44L and (o) IFIT1 RNA levels (48 hpi) after knockdown of control or MMR genes. Data shown as mean ± SD, n=4 independent samples. Data are representative of at least three independent experiments. (p) Western blot of IFIT1 in H441 cells following the specified treatments. Tubulin = loading control. For all panels: p-values calculated using unpaired two-tailed t tests; representative of two independent experiments, unless otherwise indicated.

    Journal: Nature microbiology

    Article Title: DNA mismatch repair controls the host innate response and cell fate after influenza virus infection

    doi: 10.1038/s41564-019-0509-3

    Figure Lengend Snippet: Loss of DNA MMR activity reduces the innate antiviral transcriptional response against influenza A virus. (a) NanoLuc reporter expression and (b) relative cell viability in H441 cells that have been treated with PBS or H2O2 (for 30 min). Data shown as mean ± SD, n=4 independent samples. (c) Fold change of Mx1 RNA levels in H441 cells following treatment with PBS or IFN-alpha +/− H2O2 treatment (for 30 min). Data shown as mean ± SD, n=4 independent samples. (d) Western blot for Mx1 in H441 cells following the specified treatments. Tubulin = loading control. (e) NanoLuc reporter expression and (f) relative cell viability in H441 cells following the specified treatments. Data shown as mean ± SD, n=4 independent samples. (g) Median fluorescent intensity of the ISRE-GFP reporter in 293T cells following the specified treatments. Data shown as mean ± SD, n=3 independent samples. (h) Model depicting the role of DNA MMR in preserving antiviral gene expression. (i) RNAseq data showing fold change of mRNA levels in H441 cells comparing PR8-infected cells transfected with non-targeting siRNA (black) or MSH2+MSH6 siRNA (blue) to mock-infected cells. Inset is a magnified view of all genes induced > 5-fold in PR8-infected cells treated with non-targeting siRNA. (j) Chart grouping all of the genes induced > 5-fold in PR8-infected cells based on the effect MMR knockdown has on their mRNA levels. (k) Heat map displaying the effect of MMR knockdown on ISG and antiviral genes from the group of genes displayed in j. (l-o) Fold induction of (l) IFI44L and (n) IFIT1 RNA levels after viral infection as well as the difference in infection-induced (m) IFI44L and (o) IFIT1 RNA levels (48 hpi) after knockdown of control or MMR genes. Data shown as mean ± SD, n=4 independent samples. Data are representative of at least three independent experiments. (p) Western blot of IFIT1 in H441 cells following the specified treatments. Tubulin = loading control. For all panels: p-values calculated using unpaired two-tailed t tests; representative of two independent experiments, unless otherwise indicated.

    Article Snippet: RNA was then prepped for RNAseq submission using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB), NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB), and NEBNext Multiplex Oligos for Illumina (NEB).

    Techniques: Activity Assay, Expressing, Western Blot, Preserving, Infection, Transfection, Two Tailed Test