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  • 99
    New England Biolabs small rna library prep kit
    Combined use of small <t>RNA</t> and poly(A) RNA spike-ins allow direct comparisons of small RNA-Seq and mRNA-Seq data. ( a ) Scatter plot of relative (transcripts per million) and absolute (molecules per µg total RNA) ERCC poly(A) spike-in (LifeTech) levels. Pearson’s r value is indicated, as well as a dashed line that represents a linear model derived from the plotted values. ( b ) One-dimensional scatter plots of miRNA/miRNA precursor and tasiRNA/tasiRNA precursor levels in Col-0 leaves, Col-0 flowers and dcl234 flowers. Horizontal black bars represent the median of each population. ( c ) Violin plots of miRNA/target and tasiRNA/target levels in Col-0 leaves, Col-0 flowers and dcl234 flowers. Violin plots are as described in the Fig. 2 legend. P
    Small Rna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 838 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 838 article reviews
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    99
    New England Biolabs ultra directional rna library prep kit
    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: <t>NEBNext®</t> Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of <t>RNA;</t> 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.. Color scale: Spearman correlation coefficients
    Ultra Directional Rna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3239 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    New England Biolabs ultra ii directional rna library prep kit
    NEAT1 forms triplexes at numerous genomic sites. ( A ) NEAT1 profiles in TriplexRNA-seq (DNA-IP) (red) and nuclear <t>RNA</t> (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 <t>oligos</t> 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.
    Ultra Ii Directional Rna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 137 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs ultra ii rna library prep kit
    ERCC reads to determine library preparation quality and back-calculate <t>RNA</t> input mass. (A) After normalizing to RNA input mass, reads aligning to the 92 ERCC spike-in transcripts correlate linearly with ERCC spike-in concentration across six orders of magnitude in all libraries prepared with the miniaturized protocol (R 2 E R C C m a s s ( p g ) T o t a l m a s s ( p g ) = E R C C r e a d s T o t a l r e a d s Back-calculated masses of <t>HeLa</t> libraries correlated strongly with QuBit quantification (R 2 = 0.9954).
    Ultra Ii Rna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 75 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Combined use of small RNA and poly(A) RNA spike-ins allow direct comparisons of small RNA-Seq and mRNA-Seq data. ( a ) Scatter plot of relative (transcripts per million) and absolute (molecules per µg total RNA) ERCC poly(A) spike-in (LifeTech) levels. Pearson’s r value is indicated, as well as a dashed line that represents a linear model derived from the plotted values. ( b ) One-dimensional scatter plots of miRNA/miRNA precursor and tasiRNA/tasiRNA precursor levels in Col-0 leaves, Col-0 flowers and dcl234 flowers. Horizontal black bars represent the median of each population. ( c ) Violin plots of miRNA/target and tasiRNA/target levels in Col-0 leaves, Col-0 flowers and dcl234 flowers. Violin plots are as described in the Fig. 2 legend. P

    Journal: Scientific Reports

    Article Title: Novel small RNA spike-in oligonucleotides enable absolute normalization of small RNA-Seq data

    doi: 10.1038/s41598-017-06174-3

    Figure Lengend Snippet: Combined use of small RNA and poly(A) RNA spike-ins allow direct comparisons of small RNA-Seq and mRNA-Seq data. ( a ) Scatter plot of relative (transcripts per million) and absolute (molecules per µg total RNA) ERCC poly(A) spike-in (LifeTech) levels. Pearson’s r value is indicated, as well as a dashed line that represents a linear model derived from the plotted values. ( b ) One-dimensional scatter plots of miRNA/miRNA precursor and tasiRNA/tasiRNA precursor levels in Col-0 leaves, Col-0 flowers and dcl234 flowers. Horizontal black bars represent the median of each population. ( c ) Violin plots of miRNA/target and tasiRNA/target levels in Col-0 leaves, Col-0 flowers and dcl234 flowers. Violin plots are as described in the Fig. 2 legend. P

    Article Snippet: The sRNA spike-in mix shown in Fig. was diluted two-fold and added to 500 ng of total RNA prior to polyacrylamide gel size-selection of 18–75 nt RNAs followed by sRNA cloning using the NEBnext small RNA library prep kit for Illumina (NEB).

    Techniques: RNA Sequencing Assay, Derivative Assay

    Small RNA spike-in design and use as a tool to estimate absolute small RNA levels. ( a ) Design of small RNA spike-in oligonucleotides. Key features of small RNA spike-ins are shown in different colors corresponding to the key. Molar amounts of oligonucleotides added per µg of total RNA are indicated in parentheses. ( b ) Scatter plot of relative small RNA spike-in levels (reads per million genome-matching reads) compared to absolute small RNA levels (molecules detected per µg of total RNA) in Col-0 flowers (biological replicate 1). Pearson’s r value is indicated, as well as a dashed line that represents a linear model derived from the plotted values. ( c ) Density plot of individual miRNA family levels in Col-0 flowers (biological replicate 1) in molecules detected per µg of total RNA. Vertical dashed line indicates the median number of molecules per miRNA family.

    Journal: Scientific Reports

    Article Title: Novel small RNA spike-in oligonucleotides enable absolute normalization of small RNA-Seq data

    doi: 10.1038/s41598-017-06174-3

    Figure Lengend Snippet: Small RNA spike-in design and use as a tool to estimate absolute small RNA levels. ( a ) Design of small RNA spike-in oligonucleotides. Key features of small RNA spike-ins are shown in different colors corresponding to the key. Molar amounts of oligonucleotides added per µg of total RNA are indicated in parentheses. ( b ) Scatter plot of relative small RNA spike-in levels (reads per million genome-matching reads) compared to absolute small RNA levels (molecules detected per µg of total RNA) in Col-0 flowers (biological replicate 1). Pearson’s r value is indicated, as well as a dashed line that represents a linear model derived from the plotted values. ( c ) Density plot of individual miRNA family levels in Col-0 flowers (biological replicate 1) in molecules detected per µg of total RNA. Vertical dashed line indicates the median number of molecules per miRNA family.

    Article Snippet: The sRNA spike-in mix shown in Fig. was diluted two-fold and added to 500 ng of total RNA prior to polyacrylamide gel size-selection of 18–75 nt RNAs followed by sRNA cloning using the NEBnext small RNA library prep kit for Illumina (NEB).

    Techniques: Derivative Assay

    Small RNA spike-ins enable accurate comparisons of small RNA levels. ( a and b ) Violin plots of individual miRNAs, tasiRNAs and siRNA family levels in either relative ( a ) or absolute ( b ) units. P-values were based on two-sample Kolmogorov-Smirnov tests. P

    Journal: Scientific Reports

    Article Title: Novel small RNA spike-in oligonucleotides enable absolute normalization of small RNA-Seq data

    doi: 10.1038/s41598-017-06174-3

    Figure Lengend Snippet: Small RNA spike-ins enable accurate comparisons of small RNA levels. ( a and b ) Violin plots of individual miRNAs, tasiRNAs and siRNA family levels in either relative ( a ) or absolute ( b ) units. P-values were based on two-sample Kolmogorov-Smirnov tests. P

    Article Snippet: The sRNA spike-in mix shown in Fig. was diluted two-fold and added to 500 ng of total RNA prior to polyacrylamide gel size-selection of 18–75 nt RNAs followed by sRNA cloning using the NEBnext small RNA library prep kit for Illumina (NEB).

    Techniques:

    SIRT7 is involved in pre-rRNA processing. ( a ) Knockdown of SIRT7 impairs pre-rRNA synthesis and processing in vivo . U2OS cells transfected with control (siCtrl) or SIRT7-specific siRNAs (siSIRT7) were metabolically labelled with 3 H-uridine. RNA was analysed by agarose gel electrophoresis and fluorography. The bar diagram shows quantification of the processing intermediates, values from siCtrl cells being set to 1. ( b ) In vitro processing assay. Extracts from L1210 cells were incubated with 32 P-labelled RNA comprising the 5′ETS depicted in the scheme above. 32 P-labelled RNA and cleavage products were analysed by gel electrophoresis and PhosphorImaging. See also Supplementary Fig. 3a . ( c ) 5′ETS processing is inhibited by NAM. The assay contained radiolabelled RNA (+541/+1290) and extracts from L1210 cells cultured for 6 h in the absence or presence of NAM. ( d ) Processing is enhanced by NAD + . Processing assays containing radiolabelled RNA (+541/+1290) were substituted with NAD + as indicated. ( e ) The catalytic activity of SIRT7 is required for pre-rRNA cleavage. Assays were supplemented with 15 or 30 ng of purified wildtype (WT) or mutant (H187Y) Flag-SIRT7 ( Supplementary Fig. 3b ). ( f ) Depletion of SIRT7 impairs processing. SIRT7 was depleted from L1210 cells by shRNAs (shSIRT7-1, shSIRT7-2, Supplementary Fig. 3c ). Extracts from non-infected cells (−) or cells expressing control shRNA (shCtrl) served as control (left). To rescue impaired cleavage, 15 ng of wild-type Flag-SIRT7 (WT) or mutant H187Y (HY) were added to SIRT7-depleted extracts (right). ( g ) Depletion of U3 snoRNA abolishes processing. U3 snoRNA was depleted by preincubating extracts with U3-specific antisense oligos (ASO, 50 ng μl −1 ) and 2 U of RNase H ( Supplementary Fig. 3d ). In vitro processing was performed with undepleted (−) or depleted extracts in the absence or presence of 15 ng Flag-SIRT7. Bar diagrams in c – g show quantification of the ratio of cleaved versus uncleaved transcripts, presented as mean±s.d. from three independent experiments (* P

    Journal: Nature Communications

    Article Title: SIRT7-dependent deacetylation of the U3-55k protein controls pre-rRNA processing

    doi: 10.1038/ncomms10734

    Figure Lengend Snippet: SIRT7 is involved in pre-rRNA processing. ( a ) Knockdown of SIRT7 impairs pre-rRNA synthesis and processing in vivo . U2OS cells transfected with control (siCtrl) or SIRT7-specific siRNAs (siSIRT7) were metabolically labelled with 3 H-uridine. RNA was analysed by agarose gel electrophoresis and fluorography. The bar diagram shows quantification of the processing intermediates, values from siCtrl cells being set to 1. ( b ) In vitro processing assay. Extracts from L1210 cells were incubated with 32 P-labelled RNA comprising the 5′ETS depicted in the scheme above. 32 P-labelled RNA and cleavage products were analysed by gel electrophoresis and PhosphorImaging. See also Supplementary Fig. 3a . ( c ) 5′ETS processing is inhibited by NAM. The assay contained radiolabelled RNA (+541/+1290) and extracts from L1210 cells cultured for 6 h in the absence or presence of NAM. ( d ) Processing is enhanced by NAD + . Processing assays containing radiolabelled RNA (+541/+1290) were substituted with NAD + as indicated. ( e ) The catalytic activity of SIRT7 is required for pre-rRNA cleavage. Assays were supplemented with 15 or 30 ng of purified wildtype (WT) or mutant (H187Y) Flag-SIRT7 ( Supplementary Fig. 3b ). ( f ) Depletion of SIRT7 impairs processing. SIRT7 was depleted from L1210 cells by shRNAs (shSIRT7-1, shSIRT7-2, Supplementary Fig. 3c ). Extracts from non-infected cells (−) or cells expressing control shRNA (shCtrl) served as control (left). To rescue impaired cleavage, 15 ng of wild-type Flag-SIRT7 (WT) or mutant H187Y (HY) were added to SIRT7-depleted extracts (right). ( g ) Depletion of U3 snoRNA abolishes processing. U3 snoRNA was depleted by preincubating extracts with U3-specific antisense oligos (ASO, 50 ng μl −1 ) and 2 U of RNase H ( Supplementary Fig. 3d ). In vitro processing was performed with undepleted (−) or depleted extracts in the absence or presence of 15 ng Flag-SIRT7. Bar diagrams in c – g show quantification of the ratio of cleaved versus uncleaved transcripts, presented as mean±s.d. from three independent experiments (* P

    Article Snippet: The RNA-seq libraries were created using the NEBNext Ultra RNA Library Prep kit for Illumina (E7530) with NEBNext Multiplex Oligos for Illumina (E7300).

    Techniques: In Vivo, Transfection, Metabolic Labelling, Agarose Gel Electrophoresis, In Vitro, Incubation, Nucleic Acid Electrophoresis, Cell Culture, Activity Assay, Purification, Mutagenesis, Infection, Expressing, shRNA, Allele-specific Oligonucleotide

    SIRT7 is associated with pre-rRNA and snoRNAs. ( a ) SIRT7 CLIP-seq reads mapped to a custom annotation file of a human rDNA repeat (middle) or the transcribed region (bottom). The region encoding 18S, 5.8S and 28S rRNA is highlighted. SIRT7 reads after subtraction of IgG reads were normalized to input reads ( y axis). ( b ) Gene ontology categories of SIRT7 CLIP-seq peaks. The most representative clusters are shown according to the ajusted P value (−log 10 ). ( c ) SIRT7-bound snoRNAs comprise C/D box, H/ACA box snoRNAs and scaRNAs. The number ( n ) and relative abundance (%) of each snoRNA class associated with SIRT7 is presented. ( d ) U3, SNORA73A and 73B snoRNAs are overrepresented among SIRT7-associated snoRNAs. SIRT7 reads mapped to corresponding snoRNAs are indicated as percentage of all snoRNAs identified by CLIP-seq. ( e ) Comparison of SIRT7-associated RNAs under native and denaturing conditions. His/V5-tagged SIRT7 expressed in HEK293T cells was affinity-purified on Ni-NTA-agarose under native or denaturing conditions, and associated RNAs were detected by RT–qPCR. Lysates from non-transfected HEK293T cells were used for control (Ctrl). Associated pre-RNA was monitored by RT–qPCR using primer H1 ( Supplementary Table 3 ). Bars represent means±s.d. from three experiments. See also Supplementary Fig. 2b,d . ( f ) ChIP assays showing association of endogenous SIRT7 (left panel) or transiently overexpressed Flag-SIRT7 (right panel) with the indicated gene loci in HEK293T cells. rDNA was amplified using primers H4 (coding) and H18 (IGS; Supplementary Table 3 ). Bars represent means±s.d. from three experiments. See also Supplementary Fig. 2d .

    Journal: Nature Communications

    Article Title: SIRT7-dependent deacetylation of the U3-55k protein controls pre-rRNA processing

    doi: 10.1038/ncomms10734

    Figure Lengend Snippet: SIRT7 is associated with pre-rRNA and snoRNAs. ( a ) SIRT7 CLIP-seq reads mapped to a custom annotation file of a human rDNA repeat (middle) or the transcribed region (bottom). The region encoding 18S, 5.8S and 28S rRNA is highlighted. SIRT7 reads after subtraction of IgG reads were normalized to input reads ( y axis). ( b ) Gene ontology categories of SIRT7 CLIP-seq peaks. The most representative clusters are shown according to the ajusted P value (−log 10 ). ( c ) SIRT7-bound snoRNAs comprise C/D box, H/ACA box snoRNAs and scaRNAs. The number ( n ) and relative abundance (%) of each snoRNA class associated with SIRT7 is presented. ( d ) U3, SNORA73A and 73B snoRNAs are overrepresented among SIRT7-associated snoRNAs. SIRT7 reads mapped to corresponding snoRNAs are indicated as percentage of all snoRNAs identified by CLIP-seq. ( e ) Comparison of SIRT7-associated RNAs under native and denaturing conditions. His/V5-tagged SIRT7 expressed in HEK293T cells was affinity-purified on Ni-NTA-agarose under native or denaturing conditions, and associated RNAs were detected by RT–qPCR. Lysates from non-transfected HEK293T cells were used for control (Ctrl). Associated pre-RNA was monitored by RT–qPCR using primer H1 ( Supplementary Table 3 ). Bars represent means±s.d. from three experiments. See also Supplementary Fig. 2b,d . ( f ) ChIP assays showing association of endogenous SIRT7 (left panel) or transiently overexpressed Flag-SIRT7 (right panel) with the indicated gene loci in HEK293T cells. rDNA was amplified using primers H4 (coding) and H18 (IGS; Supplementary Table 3 ). Bars represent means±s.d. from three experiments. See also Supplementary Fig. 2d .

    Article Snippet: The RNA-seq libraries were created using the NEBNext Ultra RNA Library Prep kit for Illumina (E7530) with NEBNext Multiplex Oligos for Illumina (E7300).

    Techniques: Cross-linking Immunoprecipitation, Affinity Purification, Quantitative RT-PCR, Transfection, Chromatin Immunoprecipitation, Amplification

    Pre-rRNA transcription and processing are attenuated under stress. ( a ) Northern blot of pre-rRNA and processing intermediates from HEK293T cells that were untreated, exposed to hyperosmotic stress for 90 min (hypertonic), or recovered to regular medium for 60 min (hypertonic rel.). Membranes were probed with 32 P-labelled antisense riboprobe specific to 47S pre-rRNA (5'ETS, top) or with ITS1 oligos hybridizing to pre-rRNA intermediates (middle panel). ( b ) Acetylation of U3-55k is increased on different cellular stress conditions. HEK293T cells expressing Flag-U3-55k were treated with actinomycin D (Act D, 0.1 μg ml −1 , 4 h), AICAR (0.5 mM, 12 h) or exposed to hypertonic stress. Acetylation of immunopurified Flag-U3-55k and equal loading was monitored on western blots using anti-pan-AcK and anti-Flag antibodies. ( c ) Cellular localization of SIRT7 and U3-55k on hyperosmotic stress. Images showing localization of GFP-U3-55k and SIRT7 in normal conditions and on exposure to hyperosmotic stress for 90 min. Nuclei were stained with Hoechst 33342. Scale bars, 10 μm. ( d ) Overexpression of SIRT7 alleviates processing defects on hypertonic stress. Northern blot of RNA from parental U2OS cells and from cells which stably express GFP-SIRT7 (U2OS-GFP-SIRT7) using 5′ETS and ITS1 probes as in a . ( e ) CLIP-RT–qPCR monitoring binding of Flag-U3-55k to pre-rRNA, U3 snoRNA and U2 snRNA in HEK293T cells cultured in normo-osmotic medium or exposed to hypertonic stress for 90 min. Precipitated RNA was analysed by RT–qPCR using the indicated primers. Bars represent the means±s.d. from three biological repeats (* P

    Journal: Nature Communications

    Article Title: SIRT7-dependent deacetylation of the U3-55k protein controls pre-rRNA processing

    doi: 10.1038/ncomms10734

    Figure Lengend Snippet: Pre-rRNA transcription and processing are attenuated under stress. ( a ) Northern blot of pre-rRNA and processing intermediates from HEK293T cells that were untreated, exposed to hyperosmotic stress for 90 min (hypertonic), or recovered to regular medium for 60 min (hypertonic rel.). Membranes were probed with 32 P-labelled antisense riboprobe specific to 47S pre-rRNA (5'ETS, top) or with ITS1 oligos hybridizing to pre-rRNA intermediates (middle panel). ( b ) Acetylation of U3-55k is increased on different cellular stress conditions. HEK293T cells expressing Flag-U3-55k were treated with actinomycin D (Act D, 0.1 μg ml −1 , 4 h), AICAR (0.5 mM, 12 h) or exposed to hypertonic stress. Acetylation of immunopurified Flag-U3-55k and equal loading was monitored on western blots using anti-pan-AcK and anti-Flag antibodies. ( c ) Cellular localization of SIRT7 and U3-55k on hyperosmotic stress. Images showing localization of GFP-U3-55k and SIRT7 in normal conditions and on exposure to hyperosmotic stress for 90 min. Nuclei were stained with Hoechst 33342. Scale bars, 10 μm. ( d ) Overexpression of SIRT7 alleviates processing defects on hypertonic stress. Northern blot of RNA from parental U2OS cells and from cells which stably express GFP-SIRT7 (U2OS-GFP-SIRT7) using 5′ETS and ITS1 probes as in a . ( e ) CLIP-RT–qPCR monitoring binding of Flag-U3-55k to pre-rRNA, U3 snoRNA and U2 snRNA in HEK293T cells cultured in normo-osmotic medium or exposed to hypertonic stress for 90 min. Precipitated RNA was analysed by RT–qPCR using the indicated primers. Bars represent the means±s.d. from three biological repeats (* P

    Article Snippet: The RNA-seq libraries were created using the NEBNext Ultra RNA Library Prep kit for Illumina (E7530) with NEBNext Multiplex Oligos for Illumina (E7300).

    Techniques: Northern Blot, Expressing, Activated Clotting Time Assay, Western Blot, Staining, Over Expression, Stable Transfection, Cross-linking Immunoprecipitation, Quantitative RT-PCR, Binding Assay, Cell Culture

    Epigenetic silencing of BNIP3 by an FTO-m6A-dependent mechanism. a - b BNIP3 expression was significantly up-regulated in both RNA and protein expression level in stable FTO-knockdown MDA-MB-231 cells ( a ) and MCF-7 cells ( b ). ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. c , d Knockdown of FTO promoted the cleavage of Caaspase 3 and decreased Bcl2 in MDA-MB-231 cells ( c ) and MCF-7 cells ( d ). e Knockdown of FTO promoted the m6A methylation in BNIP3 mRNA by the m6A MeRIP analysis. * P ≤ 0.05. f Wild-type or m6A consensus sequence mutant BNIP3 3’UTR was fused with firefly luciferase reporter. Mutation of m6A consensus sequences were generated by replacing adenosine with thymine. g Relative luciferase activity of the wild-type and 3 mutant BNIP3 3’UTR reporter vectors in FTO-knockdown MDA-MB-231 cells

    Journal: Molecular Cancer

    Article Title: RNA N6-methyladenosine demethylase FTO promotes breast tumor progression through inhibiting BNIP3

    doi: 10.1186/s12943-019-1004-4

    Figure Lengend Snippet: Epigenetic silencing of BNIP3 by an FTO-m6A-dependent mechanism. a - b BNIP3 expression was significantly up-regulated in both RNA and protein expression level in stable FTO-knockdown MDA-MB-231 cells ( a ) and MCF-7 cells ( b ). ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. c , d Knockdown of FTO promoted the cleavage of Caaspase 3 and decreased Bcl2 in MDA-MB-231 cells ( c ) and MCF-7 cells ( d ). e Knockdown of FTO promoted the m6A methylation in BNIP3 mRNA by the m6A MeRIP analysis. * P ≤ 0.05. f Wild-type or m6A consensus sequence mutant BNIP3 3’UTR was fused with firefly luciferase reporter. Mutation of m6A consensus sequences were generated by replacing adenosine with thymine. g Relative luciferase activity of the wild-type and 3 mutant BNIP3 3’UTR reporter vectors in FTO-knockdown MDA-MB-231 cells

    Article Snippet: For high-throughput sequencing, purified RNA fragments from m6A-MeRIP were used for library construction with the NEBNext Ultra RNA library Prep kit for Illumina (E7530S, NEB, USA) and were sequenced by Illumina HiSeq 2000.

    Techniques: Expressing, Multiple Displacement Amplification, Methylation, Sequencing, Mutagenesis, Luciferase, Generated, Activity Assay

    Up-regulation of FTO RNA demethylase in human breast cancer. a Heat map diagram of differential gene expression in breast tumors and normal tissues. b Expression of the m6A regulatory enzymes in primary human breast tumors. ** P ≤ 0.01, *** P ≤ 0.001. c Relative FTO mRNA expression level in molecular subtypes and clinical stages of breast tumors. NORM: normal tissues; TNBC: ER−/PR−/Her2-; DNBC: ER−/PR−/Her2+; TPBC: ER+/PR+/Her2+. ** P ≤ 0.01, **** P ≤ 0.0001. d Higher levels of FTO in human breast cancer tissues in comparison with normal breast tissues by immunohistochemistry assay. e FTO up-regulation was quantified from the immunohistochemistry results. f The global mRNA m6A level in human breast cancer samples determined by RNA m6A dot-blotting assay. g The global mRNA m6A level in human breast cancer samples determined by RNA m6A colorimetric analysis. * P ≤ 0.05. h FTO up-regulation was significantly associated with shorter overall survival in patients with advanced stage of breast cancer. i FTO up-regulation was significantly associated with shorter overall survival in patients with ER negative breast cancer

    Journal: Molecular Cancer

    Article Title: RNA N6-methyladenosine demethylase FTO promotes breast tumor progression through inhibiting BNIP3

    doi: 10.1186/s12943-019-1004-4

    Figure Lengend Snippet: Up-regulation of FTO RNA demethylase in human breast cancer. a Heat map diagram of differential gene expression in breast tumors and normal tissues. b Expression of the m6A regulatory enzymes in primary human breast tumors. ** P ≤ 0.01, *** P ≤ 0.001. c Relative FTO mRNA expression level in molecular subtypes and clinical stages of breast tumors. NORM: normal tissues; TNBC: ER−/PR−/Her2-; DNBC: ER−/PR−/Her2+; TPBC: ER+/PR+/Her2+. ** P ≤ 0.01, **** P ≤ 0.0001. d Higher levels of FTO in human breast cancer tissues in comparison with normal breast tissues by immunohistochemistry assay. e FTO up-regulation was quantified from the immunohistochemistry results. f The global mRNA m6A level in human breast cancer samples determined by RNA m6A dot-blotting assay. g The global mRNA m6A level in human breast cancer samples determined by RNA m6A colorimetric analysis. * P ≤ 0.05. h FTO up-regulation was significantly associated with shorter overall survival in patients with advanced stage of breast cancer. i FTO up-regulation was significantly associated with shorter overall survival in patients with ER negative breast cancer

    Article Snippet: For high-throughput sequencing, purified RNA fragments from m6A-MeRIP were used for library construction with the NEBNext Ultra RNA library Prep kit for Illumina (E7530S, NEB, USA) and were sequenced by Illumina HiSeq 2000.

    Techniques: Expressing, Immunohistochemistry

    RNA-Seq and m6A-Seq identified BNIP3 as a downstream target of FTO-mediated m6A modification. a Venn diagram illustrated overlap in differentially expressed genes in FTO-knockdown MDA-MB-231 cells and MCF-4 cells treated with DMOG. b KEGG analysis shows that FTO-knockdown regulate pathways involved in cell proliferation, cell cycle and apoptosis. c m6A-Seq identification of m6A modification in BNIP3 mRNA near to the YTHDF2 binding sites. d Differentially expressed genes by inhibiting or knockdown of FTO involved in the FoxO signaling pathway. Red color indicates up-regulated genes, while purple color indicates down-regulated genes. e , f Heatmap of up-regulated genes in FTO-knockdown MDA-MB-231 cells ( e ) and MCF-4 cells treated with DMOG ( f ). g Co-expression analysis of BNIP3 by the string

    Journal: Molecular Cancer

    Article Title: RNA N6-methyladenosine demethylase FTO promotes breast tumor progression through inhibiting BNIP3

    doi: 10.1186/s12943-019-1004-4

    Figure Lengend Snippet: RNA-Seq and m6A-Seq identified BNIP3 as a downstream target of FTO-mediated m6A modification. a Venn diagram illustrated overlap in differentially expressed genes in FTO-knockdown MDA-MB-231 cells and MCF-4 cells treated with DMOG. b KEGG analysis shows that FTO-knockdown regulate pathways involved in cell proliferation, cell cycle and apoptosis. c m6A-Seq identification of m6A modification in BNIP3 mRNA near to the YTHDF2 binding sites. d Differentially expressed genes by inhibiting or knockdown of FTO involved in the FoxO signaling pathway. Red color indicates up-regulated genes, while purple color indicates down-regulated genes. e , f Heatmap of up-regulated genes in FTO-knockdown MDA-MB-231 cells ( e ) and MCF-4 cells treated with DMOG ( f ). g Co-expression analysis of BNIP3 by the string

    Article Snippet: For high-throughput sequencing, purified RNA fragments from m6A-MeRIP were used for library construction with the NEBNext Ultra RNA library Prep kit for Illumina (E7530S, NEB, USA) and were sequenced by Illumina HiSeq 2000.

    Techniques: RNA Sequencing Assay, Modification, Multiple Displacement Amplification, Binding Assay, Expressing

    Evolution and performance of nucleocapsids with exterior surface mutations in vitro or in vivo a . Heatmap of log enrichments between the injected pool and RNA recovered from the tail vein 60 minutes later. Purple and orange indicate mutations that were depleted or enriched in the selected population, respectively. Blue squares and black dots indicate the I53-50-v3 starting sequence and I53-50-v4 selected sequence, respectively. Residues not in the designed combinatorial library are colored gray. Note the strong enrichment of the E67K mutation and corresponding depletion of the native E67 allele. b . Design model of I53-50-v4. Coloring is as described in . c . Four variants were tested: a consensus sequence based on the most common residue at each position after selection in murine circulation (Consensus, I53-50-v4), the full length sequence with the greatest fold increase in population fraction (Most_enriched), the sequence with the most total counts (Top_count), and I53-50-v3 with only the E67K mutation (v3_E67K). Previous versions (I53-50-v1 through I53-50-v3) were also included as benchmarks. Each variant was individually expressed and purified by IMAC before being pooled (equal protein concentration) and purified en masse by SEC. The resulting nucleocapsid pool was then incubated in whole blood (n = 3 independent reactions). RNA was recovered at the indicated time points, and the fraction of each variant was determined by Illumina MiSeq counts taken at each time point. d . The same nucleocapsid pool used in ( c ) was injected retro-orbitally into mice (n = 5 biologically independent mice). I53-50-v3 was evaluated with (v3) and without (v3H) the H6Q and H9Q mutations, and both variants were found to have similar behavior. Error bars represent standard error of the mean. Fig. 1a

    Journal:

    Article Title: Evolution of a Designed Protein Assembly Encapsulating its Own RNA Genome

    doi: 10.1038/nature25157

    Figure Lengend Snippet: Evolution and performance of nucleocapsids with exterior surface mutations in vitro or in vivo a . Heatmap of log enrichments between the injected pool and RNA recovered from the tail vein 60 minutes later. Purple and orange indicate mutations that were depleted or enriched in the selected population, respectively. Blue squares and black dots indicate the I53-50-v3 starting sequence and I53-50-v4 selected sequence, respectively. Residues not in the designed combinatorial library are colored gray. Note the strong enrichment of the E67K mutation and corresponding depletion of the native E67 allele. b . Design model of I53-50-v4. Coloring is as described in . c . Four variants were tested: a consensus sequence based on the most common residue at each position after selection in murine circulation (Consensus, I53-50-v4), the full length sequence with the greatest fold increase in population fraction (Most_enriched), the sequence with the most total counts (Top_count), and I53-50-v3 with only the E67K mutation (v3_E67K). Previous versions (I53-50-v1 through I53-50-v3) were also included as benchmarks. Each variant was individually expressed and purified by IMAC before being pooled (equal protein concentration) and purified en masse by SEC. The resulting nucleocapsid pool was then incubated in whole blood (n = 3 independent reactions). RNA was recovered at the indicated time points, and the fraction of each variant was determined by Illumina MiSeq counts taken at each time point. d . The same nucleocapsid pool used in ( c ) was injected retro-orbitally into mice (n = 5 biologically independent mice). I53-50-v3 was evaluated with (v3) and without (v3H) the H6Q and H9Q mutations, and both variants were found to have similar behavior. Error bars represent standard error of the mean. Fig. 1a

    Article Snippet: The purified RNA was quantitated using a Qubit RNA HS Assay Kit, and 100 ng of RNA was used to prepare each RNAseq library with a NEBNext® Ultra™ RNA Library Prep Kit for Illumina® kit (NEB, E7530S).

    Techniques: In Vitro, In Vivo, Injection, Sequencing, Mutagenesis, Selection, Variant Assay, Purification, Protein Concentration, Size-exclusion Chromatography, Incubation, Mouse Assay

    Evolution and performance of nucleocapsids modified with hydrophilic polypeptides in vitro or in vivo a . The change in population fraction corresponding to each variant was calculated from Illumina MiSeq counts for the input pool (t = 0), RNA recovered from circulation after 30 minutes (n = 3 biologically independent mice), and RNA recovered from circulation after 60 minutes (n = 2 biologically independent mice). b . Scatter plot of log 10 enrichment of each hydrophilic polypeptide versus its net charge as calculated from the total number of charged residues in its sequence. c . Scatter plot of log 10 enrichment of each polypeptide versus the number of unique amino acids in its sequence. d . Each of 11 variants were individually expressed and purified by IMAC before being pooled (equal protein concentration) and purified en masse by SEC. The resulting nucleocapsid pool was then incubated in heparinized whole blood at 37 ° C (n = 3 independent reactions per time point). RNA was recovered at the indicated time points, and the fraction of each variant was determined by Illumina MiSeq counts taken at each time point. e . The same nucleocapsid pool used in ( d ) was injected retro-orbitally into mice (n = 5 biologically independent mice). RNA content was then assessed as in ( d ) using RNA isolated from tail vein draws at the indicated time points. All variants exhibit high stability in blood; however, the unmodified I53-50-v3 nucleocapsid (no polypeptide, blue) and a negative control polypeptide (ESESG, red) are cleared rapidly from circulation in vivo . Error bars represent standard error of the mean.

    Journal:

    Article Title: Evolution of a Designed Protein Assembly Encapsulating its Own RNA Genome

    doi: 10.1038/nature25157

    Figure Lengend Snippet: Evolution and performance of nucleocapsids modified with hydrophilic polypeptides in vitro or in vivo a . The change in population fraction corresponding to each variant was calculated from Illumina MiSeq counts for the input pool (t = 0), RNA recovered from circulation after 30 minutes (n = 3 biologically independent mice), and RNA recovered from circulation after 60 minutes (n = 2 biologically independent mice). b . Scatter plot of log 10 enrichment of each hydrophilic polypeptide versus its net charge as calculated from the total number of charged residues in its sequence. c . Scatter plot of log 10 enrichment of each polypeptide versus the number of unique amino acids in its sequence. d . Each of 11 variants were individually expressed and purified by IMAC before being pooled (equal protein concentration) and purified en masse by SEC. The resulting nucleocapsid pool was then incubated in heparinized whole blood at 37 ° C (n = 3 independent reactions per time point). RNA was recovered at the indicated time points, and the fraction of each variant was determined by Illumina MiSeq counts taken at each time point. e . The same nucleocapsid pool used in ( d ) was injected retro-orbitally into mice (n = 5 biologically independent mice). RNA content was then assessed as in ( d ) using RNA isolated from tail vein draws at the indicated time points. All variants exhibit high stability in blood; however, the unmodified I53-50-v3 nucleocapsid (no polypeptide, blue) and a negative control polypeptide (ESESG, red) are cleared rapidly from circulation in vivo . Error bars represent standard error of the mean.

    Article Snippet: The purified RNA was quantitated using a Qubit RNA HS Assay Kit, and 100 ng of RNA was used to prepare each RNAseq library with a NEBNext® Ultra™ RNA Library Prep Kit for Illumina® kit (NEB, E7530S).

    Techniques: Modification, In Vitro, In Vivo, Variant Assay, Mouse Assay, Sequencing, Purification, Protein Concentration, Size-exclusion Chromatography, Incubation, Injection, Isolation, Negative Control

    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: 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.. 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: 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.. Color scale: Spearman correlation coefficients

    Article Snippet: NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina with 4 ng of 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: NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina with 4 ng of 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: NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina with 4 ng of 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: NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina with 4 ng of RNA

    Techniques:

    Descriptive characteristics of raw and mapped reads. a Total number of raw reads and number of reads mapped to the mouse genome (mm10, GRCm38.84). b Percentage of reads mapped to exonic, intronic and intergenic regions. 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

    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 characteristics of raw and mapped reads. a Total number of raw reads and number of reads mapped to the mouse genome (mm10, GRCm38.84). b Percentage of reads mapped to exonic, intronic and intergenic regions. 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

    Article Snippet: NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina with 4 ng of 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: NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina with 4 ng of RNA

    Techniques: Expressing

    Venn diagrams of identified features in the different libraries. The features with CPM ≥ 1 in at least one out of 3 replicates were used to generate these plots. a and c represent input samples and b and d represent IP samples. Most transcripts were detected by all kits tested. However, a higher rate of agreement is seen between the NEB, TruSeq and SMART-Seq prepared 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

    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: Venn diagrams of identified features in the different libraries. The features with CPM ≥ 1 in at least one out of 3 replicates were used to generate these plots. a and c represent input samples and b and d represent IP samples. Most transcripts were detected by all kits tested. However, a higher rate of agreement is seen between the NEB, TruSeq and SMART-Seq prepared 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

    Article Snippet: NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina with 4 ng of RNA

    Techniques:

    piRNA-mediated chromatin changes regulate P -element splicing a , Diagram of IVS3 transgenic reporter 20 : white box, germline-specific hsp83 promoter; ATG, codon start; gray box, nuclear localization signal; purple, P -element fragment containing IVS3 (inverted triangle) and exonic flanking sequences (boxes); blue box, LacZ ; green box, neo . b , RT-qPCR analysis using adult ovaries of [F1] progeny originated from reciprocal crosses between Harwich strain and w 1118 flies carrying the IVS3 reporter. Bars represent the percentage of spliced IVS3 reporter transgenic transcripts, determined by the ratio between IVS3 spliced transgenic RNA (quantified using primers that specifically anneal to spliced transgenic transcripts) and total IVS3 reporter transgenic mRNA (quantified using primers that anneal within the LacZ coding sequence). Results are presented as means of spliced transgenic RNA (percentage) ± standard deviation (n=2 independent biological replicate experiments). c , RT-qPCR analysis in aub /+ heterozygous, aub mutant, vas /+ heterozygous, vas mutant, panx /+ heterozygous, and panx mutant, carrying the transgenic IVS3 reporter. All experiments were performed in a Harwich background (n > =2 independent biological replicate experiments). Results are represented as in ( b ). d , RT-qPCR analysis on adult ovaries of germline knockdowns (KD) targeting piRNA pathway components involved in chromatin targeting ( piwi , arx/Gstf1 , Panx/Silencio, Su(var)205/HP1a , and mael ). Results are presented as means of fold changes in germline KDs in relation to controls ( white or mCherry germline KDs) ± standard deviation. All analyses were performed in a Harwich background (n > =2 independent biological replicate experiments). e-f , Genome browser view of two of the P -element insertions showing transcriptional activity. Normalized RNA-seq and H3K9me3 ChIP signals are presented in gray and blue, respectively. The gray bar crossing the plots represents P -element chromosomal insertion site. Annotation is at bottom: purple boxes, coding exons; pink boxes, untranslated regions (UTR); purple lines, introns; gray box, P -element insertion. View showing P -element insertion into Bacc/CG9894 ( e ) and Lk6 ( f ) genes.

    Journal: Nature

    Article Title: piRNA-mediated regulation of transposon alternative splicing in soma and germline

    doi: 10.1038/nature25018

    Figure Lengend Snippet: piRNA-mediated chromatin changes regulate P -element splicing a , Diagram of IVS3 transgenic reporter 20 : white box, germline-specific hsp83 promoter; ATG, codon start; gray box, nuclear localization signal; purple, P -element fragment containing IVS3 (inverted triangle) and exonic flanking sequences (boxes); blue box, LacZ ; green box, neo . b , RT-qPCR analysis using adult ovaries of [F1] progeny originated from reciprocal crosses between Harwich strain and w 1118 flies carrying the IVS3 reporter. Bars represent the percentage of spliced IVS3 reporter transgenic transcripts, determined by the ratio between IVS3 spliced transgenic RNA (quantified using primers that specifically anneal to spliced transgenic transcripts) and total IVS3 reporter transgenic mRNA (quantified using primers that anneal within the LacZ coding sequence). Results are presented as means of spliced transgenic RNA (percentage) ± standard deviation (n=2 independent biological replicate experiments). c , RT-qPCR analysis in aub /+ heterozygous, aub mutant, vas /+ heterozygous, vas mutant, panx /+ heterozygous, and panx mutant, carrying the transgenic IVS3 reporter. All experiments were performed in a Harwich background (n > =2 independent biological replicate experiments). Results are represented as in ( b ). d , RT-qPCR analysis on adult ovaries of germline knockdowns (KD) targeting piRNA pathway components involved in chromatin targeting ( piwi , arx/Gstf1 , Panx/Silencio, Su(var)205/HP1a , and mael ). Results are presented as means of fold changes in germline KDs in relation to controls ( white or mCherry germline KDs) ± standard deviation. All analyses were performed in a Harwich background (n > =2 independent biological replicate experiments). e-f , Genome browser view of two of the P -element insertions showing transcriptional activity. Normalized RNA-seq and H3K9me3 ChIP signals are presented in gray and blue, respectively. The gray bar crossing the plots represents P -element chromosomal insertion site. Annotation is at bottom: purple boxes, coding exons; pink boxes, untranslated regions (UTR); purple lines, introns; gray box, P -element insertion. View showing P -element insertion into Bacc/CG9894 ( e ) and Lk6 ( f ) genes.

    Article Snippet: Poly(A)-selected RNA-sequencing (RNA-seq) analysis was performed on 2.5μg of RNA purified from adult ovaries using the NEBNext® Poly(A) mRNA Magnetic Isolation Module and the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® .

    Techniques: Transgenic Assay, Quantitative RT-PCR, Sequencing, Standard Deviation, Mutagenesis, Activity Assay, RNA Sequencing Assay, Chromatin Immunoprecipitation

    P -element mRNA steady-state levels do not change in piRNA mutants in comparison to respective heterozygous a-b , Scatterplots showing the expression of genes (gray dots) and transposons (blue dots), as measured by RNA-seq analysis (expressed in fragments per kilobase per million fragments, FPKM, log 10 ), in aub /+ heterozygous vs. aub mutant adult ovaries ( a ) and piwi /+ heterozygous vs. piwi mutant adult ovaries ( b ) comparisons. P -element expression is shown in green. Transposons with RNA abundance changes > 10-fold are outlined in red. Experiments were repeated two times with similar results. c-d, Genome-wide analysis of splicing changes in aub /+ heterozygous vs. aub mutant adult ovaries ( c ) and piwi /+ heterozygous vs. piwi mutant adult ovaries ( d ) comparisons. Quantification of splicing changes was performed using RNA-seq data and the JUM method 44,45 . Results are expressed as log 2 fold changes in splicing (mutant/heterozygous). Gray dots represent individual splice junctions identified, sorted by fold change values. Green dots represent splice junctions with statistically significant changes in heterozygous vs. mutant comparisons (adjust p -value

    Journal: Nature

    Article Title: piRNA-mediated regulation of transposon alternative splicing in soma and germline

    doi: 10.1038/nature25018

    Figure Lengend Snippet: P -element mRNA steady-state levels do not change in piRNA mutants in comparison to respective heterozygous a-b , Scatterplots showing the expression of genes (gray dots) and transposons (blue dots), as measured by RNA-seq analysis (expressed in fragments per kilobase per million fragments, FPKM, log 10 ), in aub /+ heterozygous vs. aub mutant adult ovaries ( a ) and piwi /+ heterozygous vs. piwi mutant adult ovaries ( b ) comparisons. P -element expression is shown in green. Transposons with RNA abundance changes > 10-fold are outlined in red. Experiments were repeated two times with similar results. c-d, Genome-wide analysis of splicing changes in aub /+ heterozygous vs. aub mutant adult ovaries ( c ) and piwi /+ heterozygous vs. piwi mutant adult ovaries ( d ) comparisons. Quantification of splicing changes was performed using RNA-seq data and the JUM method 44,45 . Results are expressed as log 2 fold changes in splicing (mutant/heterozygous). Gray dots represent individual splice junctions identified, sorted by fold change values. Green dots represent splice junctions with statistically significant changes in heterozygous vs. mutant comparisons (adjust p -value

    Article Snippet: Poly(A)-selected RNA-sequencing (RNA-seq) analysis was performed on 2.5μg of RNA purified from adult ovaries using the NEBNext® Poly(A) mRNA Magnetic Isolation Module and the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® .

    Techniques: Expressing, RNA Sequencing Assay, Mutagenesis, Genome Wide

    piRNA-mediated chromatin targeting machinery modulates splicing of the Gypsy retrotransposon in Ovarian Somatic Cells (OSC) a , Diagram of Gypsy retrotransposon: gray boxes, long terminal repeats; white boxes, coding sequences; red inverted triangle, splicing that generates env mRNA. b , RNA-seq signal (RPM) at the Gypsy splicing donor and acceptor sites in representative control (GFP KD) and piwi KD conditions. Raw data sets from Sienski et al, 2015. Experiments were repeated three times with similar results. c , Percentage of splicing for Gypsy splicing donor and acceptor sites as determined by RNA-seq analysis performed in OSC cells KDs of piRNA machinery components. Bars represent the number of split-reads for the env splicing donor and acceptor junctions normalized to the total number of sense Gypsy reads mapping to the same junction. Results are represented as means. With the exception arx/Gst1, Su(var)205/HP1a, mael, and H1 7,25 , experiments were repeated two or more times. Raw data sets from Ohtani et al, 2013 7 ; Sienski et al, 2015 9 ; Iwasaki et al, 2016 25 .

    Journal: Nature

    Article Title: piRNA-mediated regulation of transposon alternative splicing in soma and germline

    doi: 10.1038/nature25018

    Figure Lengend Snippet: piRNA-mediated chromatin targeting machinery modulates splicing of the Gypsy retrotransposon in Ovarian Somatic Cells (OSC) a , Diagram of Gypsy retrotransposon: gray boxes, long terminal repeats; white boxes, coding sequences; red inverted triangle, splicing that generates env mRNA. b , RNA-seq signal (RPM) at the Gypsy splicing donor and acceptor sites in representative control (GFP KD) and piwi KD conditions. Raw data sets from Sienski et al, 2015. Experiments were repeated three times with similar results. c , Percentage of splicing for Gypsy splicing donor and acceptor sites as determined by RNA-seq analysis performed in OSC cells KDs of piRNA machinery components. Bars represent the number of split-reads for the env splicing donor and acceptor junctions normalized to the total number of sense Gypsy reads mapping to the same junction. Results are represented as means. With the exception arx/Gst1, Su(var)205/HP1a, mael, and H1 7,25 , experiments were repeated two or more times. Raw data sets from Ohtani et al, 2013 7 ; Sienski et al, 2015 9 ; Iwasaki et al, 2016 25 .

    Article Snippet: Poly(A)-selected RNA-sequencing (RNA-seq) analysis was performed on 2.5μg of RNA purified from adult ovaries using the NEBNext® Poly(A) mRNA Magnetic Isolation Module and the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® .

    Techniques: RNA Sequencing Assay

    Analysis of P -element expression and splicing in adult ovaries of non-dysgenic and dysgenic progeny grown at 18°C a , Scatterplot showing the expression of genes (gray dots) and transposons (blue dots), as measured by RNA-seq analysis (expressed in fragments per kilobase per million fragments, FPKM, log 10 ), in adult ovaries of non-dysgenic vs. dysgenic progeny grown at 18°C. P -element expression is shown in green. Genes containing P-element insertion in Harwich strain are depicted in purple. b-c , Percentage of splicing for P -element IVS1, IVS2 ( b ), and IVS3 ( c ) splicing junctions as determined by RNA-seq analysis in non-dysgenic (green) and dysgenic (red) adult ovaries. Bars represent percentage of splicing, calculated as the number of split-reads for each splicing junction normalized to the total number of reads mapping to the same junction. Results are represented as means ± standard deviation (n=2 independent biological replicate experiments). d , Ethidium bromide-stained gel displaying RT-PCR reactions with primers flanking the P -element IVS3 intron in adult ovaries of non-dysgenic and dysgenic progeny grown at 18°C, as well as aub /+ heterozygous and aub mutant grown at 29°C. Size scale in base pairs (bp) is presented for each gel. As shown, experiments were repeated three times with similar results. For gel source data, see Supplementary Figure 1 . e , RT-qPCR analysis testing accumulation of IVS3 spliced mRNA on non-dysgenic and dysgenic progeny (ovaries) grown at 18°C. Results are expressed as means of percentage of expression relative to controls ± standard deviation (n=3 independent biological replicate experiments). f, Genome-wide analysis of splicing changes in in adult ovaries of non-dysgenic vs. dysgenic progeny grown at 18°C. Quantification of splicing changes was performed using RNA-seq data and the JUM method 44,45 . Results are expressed as log 2 fold changes in splicing (dysgenic/non-dysgenic). Gray dots represent individual splice junctions identified, sorted by fold change values. Green dots represent splice junctions with statistically significant changes in heterozygous vs. mutant comparisons (adjust p -value

    Journal: Nature

    Article Title: piRNA-mediated regulation of transposon alternative splicing in soma and germline

    doi: 10.1038/nature25018

    Figure Lengend Snippet: Analysis of P -element expression and splicing in adult ovaries of non-dysgenic and dysgenic progeny grown at 18°C a , Scatterplot showing the expression of genes (gray dots) and transposons (blue dots), as measured by RNA-seq analysis (expressed in fragments per kilobase per million fragments, FPKM, log 10 ), in adult ovaries of non-dysgenic vs. dysgenic progeny grown at 18°C. P -element expression is shown in green. Genes containing P-element insertion in Harwich strain are depicted in purple. b-c , Percentage of splicing for P -element IVS1, IVS2 ( b ), and IVS3 ( c ) splicing junctions as determined by RNA-seq analysis in non-dysgenic (green) and dysgenic (red) adult ovaries. Bars represent percentage of splicing, calculated as the number of split-reads for each splicing junction normalized to the total number of reads mapping to the same junction. Results are represented as means ± standard deviation (n=2 independent biological replicate experiments). d , Ethidium bromide-stained gel displaying RT-PCR reactions with primers flanking the P -element IVS3 intron in adult ovaries of non-dysgenic and dysgenic progeny grown at 18°C, as well as aub /+ heterozygous and aub mutant grown at 29°C. Size scale in base pairs (bp) is presented for each gel. As shown, experiments were repeated three times with similar results. For gel source data, see Supplementary Figure 1 . e , RT-qPCR analysis testing accumulation of IVS3 spliced mRNA on non-dysgenic and dysgenic progeny (ovaries) grown at 18°C. Results are expressed as means of percentage of expression relative to controls ± standard deviation (n=3 independent biological replicate experiments). f, Genome-wide analysis of splicing changes in in adult ovaries of non-dysgenic vs. dysgenic progeny grown at 18°C. Quantification of splicing changes was performed using RNA-seq data and the JUM method 44,45 . Results are expressed as log 2 fold changes in splicing (dysgenic/non-dysgenic). Gray dots represent individual splice junctions identified, sorted by fold change values. Green dots represent splice junctions with statistically significant changes in heterozygous vs. mutant comparisons (adjust p -value

    Article Snippet: Poly(A)-selected RNA-sequencing (RNA-seq) analysis was performed on 2.5μg of RNA purified from adult ovaries using the NEBNext® Poly(A) mRNA Magnetic Isolation Module and the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® .

    Techniques: Expressing, RNA Sequencing Assay, Standard Deviation, Staining, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Quantitative RT-PCR, Genome Wide

    piRNAs, but not siRNAs, modulate P -element splicing in germ cells All analyses were performed in a Harwich background. a , RT-qPCR analysis on piRNA- ( piwi , aub , ago3 , spnE , and vas ) and siRNA-biogenesis ( dcr2 and ago2 ) mutant adult ovaries. Results are presented as means of fold changes in mutants in relation to respective heterozygote ± standard deviation (n > =2 independent biological replicate experiments). b , Ethidium bromide-stained gel displaying RT-PCR reactions with primers flanking the P -element IVS3 intron in piRNA- and siRNA-biogenesis mutants. Size scale in base pairs (bp) is presented for each gel. Experiments were repeated two times with similar results. For gel source data, see Supplementary Figure 1 . c , Density plots for normalized strand-specific mRNA steady-state levels (measured by RNA-seq and represented as reads per million, RPM) over consensus P -element sequence (top diagram) in aub /+ heterozygous (yellow, top plot) and aub mutant (blue, bottom plot) adult ovaries. The number and position of split-reads (represented by arcs that connect exons) observed for IVS1, IVS2, and IVS3 splicing junctions is shown below each density plot. Experiments were repeated two times with similar results. d-e , Percentage of splicing for P -element IVS1, IVS2 ( d ), and IVS3 ( e ). Splicing was quantified using RNA-seq analysis in aub /+ heterozygous (yellow), aub mutant (blue), piwi /+ heterozygous (beige), and piwi mutant (purple) adult ovaries. Percentage of splicing was calculated as the number of split-reads for each splicing junction normalized to the total number of reads mapping to the same junction. Results are represented as means ± standard deviation (n=2 independent biological replicate experiments). f , Representative confocal projections of RNA-FISH signal (grayscale) showing the accumulation of sense RNA for Burdock and P -element transposons in heterozygous and mutant egg chambers. Bottom panels depict projections of representative nurse cell nuclei (purple dotted line) for the same genotypes. Scale bars, 20 μM. Experiments were repeated two or more times with similar results.

    Journal: Nature

    Article Title: piRNA-mediated regulation of transposon alternative splicing in soma and germline

    doi: 10.1038/nature25018

    Figure Lengend Snippet: piRNAs, but not siRNAs, modulate P -element splicing in germ cells All analyses were performed in a Harwich background. a , RT-qPCR analysis on piRNA- ( piwi , aub , ago3 , spnE , and vas ) and siRNA-biogenesis ( dcr2 and ago2 ) mutant adult ovaries. Results are presented as means of fold changes in mutants in relation to respective heterozygote ± standard deviation (n > =2 independent biological replicate experiments). b , Ethidium bromide-stained gel displaying RT-PCR reactions with primers flanking the P -element IVS3 intron in piRNA- and siRNA-biogenesis mutants. Size scale in base pairs (bp) is presented for each gel. Experiments were repeated two times with similar results. For gel source data, see Supplementary Figure 1 . c , Density plots for normalized strand-specific mRNA steady-state levels (measured by RNA-seq and represented as reads per million, RPM) over consensus P -element sequence (top diagram) in aub /+ heterozygous (yellow, top plot) and aub mutant (blue, bottom plot) adult ovaries. The number and position of split-reads (represented by arcs that connect exons) observed for IVS1, IVS2, and IVS3 splicing junctions is shown below each density plot. Experiments were repeated two times with similar results. d-e , Percentage of splicing for P -element IVS1, IVS2 ( d ), and IVS3 ( e ). Splicing was quantified using RNA-seq analysis in aub /+ heterozygous (yellow), aub mutant (blue), piwi /+ heterozygous (beige), and piwi mutant (purple) adult ovaries. Percentage of splicing was calculated as the number of split-reads for each splicing junction normalized to the total number of reads mapping to the same junction. Results are represented as means ± standard deviation (n=2 independent biological replicate experiments). f , Representative confocal projections of RNA-FISH signal (grayscale) showing the accumulation of sense RNA for Burdock and P -element transposons in heterozygous and mutant egg chambers. Bottom panels depict projections of representative nurse cell nuclei (purple dotted line) for the same genotypes. Scale bars, 20 μM. Experiments were repeated two or more times with similar results.

    Article Snippet: Poly(A)-selected RNA-sequencing (RNA-seq) analysis was performed on 2.5μg of RNA purified from adult ovaries using the NEBNext® Poly(A) mRNA Magnetic Isolation Module and the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® .

    Techniques: Quantitative RT-PCR, Mutagenesis, Standard Deviation, Staining, Reverse Transcription Polymerase Chain Reaction, RNA Sequencing Assay, Sequencing, Fluorescence In Situ Hybridization

    RtcB re-ligates RNA43 to purified 70S Δ43 ribosomes in vitro . ( A ) Schematic showing the in vitro ribosome repair assay (see text for details). ( B ) RNA43 AgeI used in the assay. Nucleotides changed in helix 45 and the binding site for primers Y12 (gray) and H17 (green) are indicated. ( C ) RT-PCR performed on RNA purified from 70S Δ43 ribosomes incubated in the absence of RtcB and RNA43 AgeI (lane 1), in the presence of either RNA43 AgeI (lane 2) or RtcB (lane 3), or both (lane 4) employing primer pairs specific for the central region of 16S rRNA (S7/X15; panel a), the 3΄end of 16S rRNA (S7/Y12; panel b), and specific for the AU nucleotide change present in 16S AgeI rRNA upon successful ligation (S7/H17; panel c). ( D ) AgeI restriction analysis and ( E ), sequencing of the RT-PCR product obtained with primer pair S7/H17 (shown in C, panel c, lane 4). Sequencing analyses from position 1509–1517 of the 16S rRNA of (a) rrsB , and (b) the RT-PCR product are shown. Nucleotides changed in RNA43 AgeI are underlined.

    Journal: Nucleic Acids Research

    Article Title: The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli

    doi: 10.1093/nar/gkw1018

    Figure Lengend Snippet: RtcB re-ligates RNA43 to purified 70S Δ43 ribosomes in vitro . ( A ) Schematic showing the in vitro ribosome repair assay (see text for details). ( B ) RNA43 AgeI used in the assay. Nucleotides changed in helix 45 and the binding site for primers Y12 (gray) and H17 (green) are indicated. ( C ) RT-PCR performed on RNA purified from 70S Δ43 ribosomes incubated in the absence of RtcB and RNA43 AgeI (lane 1), in the presence of either RNA43 AgeI (lane 2) or RtcB (lane 3), or both (lane 4) employing primer pairs specific for the central region of 16S rRNA (S7/X15; panel a), the 3΄end of 16S rRNA (S7/Y12; panel b), and specific for the AU nucleotide change present in 16S AgeI rRNA upon successful ligation (S7/H17; panel c). ( D ) AgeI restriction analysis and ( E ), sequencing of the RT-PCR product obtained with primer pair S7/H17 (shown in C, panel c, lane 4). Sequencing analyses from position 1509–1517 of the 16S rRNA of (a) rrsB , and (b) the RT-PCR product are shown. Nucleotides changed in RNA43 AgeI are underlined.

    Article Snippet: RNA sample preparation (total RNA and polysomal RNA) from E. coli strain MC4100 F΄ without or with plasmid pSA1 15 min upon induction of mazF expression was described elsewhere ( ). cDNA libraries were prepared using 50–100 ng of the rRNA-depleted RNA with NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, E7420), following the manufacturer's instructions and sequenced on Illumina HiSequ2000 (read length 100bp).

    Techniques: Purification, In Vitro, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Incubation, Ligation, Sequencing

    RNA43, 16S Δ43 rRNA and 70S Δ43 ribosomes are stable during stress conditions. ( A ) Schematic depiction of the experimental approach to assess the stability of RNA43 and 16S Δ43 rRNA in vivo . A schematic growth curve in the absence of IPTG is shown in black. At OD 600 of 0.3 the culture was divided and IPTG was added to one half to induce mazF expression (growth is blocked as indicated in red). 60 minutes thereafter, cells were washed and resuspended in fresh medium comprising rifampicin (in blue). ( B ) Samples withdrawn at the time points indicated were subjected to northern blot analysis with probes specific for the central domain of the 16S rRNA (CD, panel a), the 3΄-terminus of the 16S rRNA (A20; panel b) and the RNA43 (A20; panel c). 5S rRNA was used as internal standard for quantification (panel d). In vitro transcribed 16S rRNA (lane 1) and RNA43 (lane 2) served as size markers. The experiment was performed in triplicate and one representative autoradiograph is shown. ( C ) Schematic of the experimental approach to assess the stability of 70S Δ43 ribosomes in vivo as described in A. ( D ) S30 extracts were prepared before (dotted lines) and 120 min after addition of rifampicin (solid lines) to untreated cells (black lines) or 60 min after induction of mazF expression (red lines) and subjected to sucrose density gradient analysis. Peaks representing 30S and 50S subunits and 70S ribosomes are indicated. The peak areas of the 30S and 50S subunits (filled areas) and 70S monosomes (hatched area) that were quantified to determine the subunits/monosome ratios are indicated. ( E ) To monitor MazF-mediated processing of the 16S rRNA, RNA was isolated from the fractions comprising the 70S monosomes. RT-PCR analysis using primers S7/X15 (upper panel), specific for both intact 16S rRNA and 16S Δ43 rRNA, and primers S7/Y12 (lower panel), yielding a product only with uncleaved 16S rRNA. NTC: no template control. Below, the binding sites of the primers are given schematically. ( F ) Ribosome sedimentation profiles of cell extracts 30 min after induction of mazF expression. Total RNA was purified from the indicated fractions (top, 30S, 50S, 70S and polysomes), respectively, and tested for the presence of RNA43 by northern blotting. Total RNA purified from the S30 extract withdrawn 30 min upon induction of mazF expression (lane 1), and in vitro transcribed RNA43 (lane 7) served as controls.

    Journal: Nucleic Acids Research

    Article Title: The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli

    doi: 10.1093/nar/gkw1018

    Figure Lengend Snippet: RNA43, 16S Δ43 rRNA and 70S Δ43 ribosomes are stable during stress conditions. ( A ) Schematic depiction of the experimental approach to assess the stability of RNA43 and 16S Δ43 rRNA in vivo . A schematic growth curve in the absence of IPTG is shown in black. At OD 600 of 0.3 the culture was divided and IPTG was added to one half to induce mazF expression (growth is blocked as indicated in red). 60 minutes thereafter, cells were washed and resuspended in fresh medium comprising rifampicin (in blue). ( B ) Samples withdrawn at the time points indicated were subjected to northern blot analysis with probes specific for the central domain of the 16S rRNA (CD, panel a), the 3΄-terminus of the 16S rRNA (A20; panel b) and the RNA43 (A20; panel c). 5S rRNA was used as internal standard for quantification (panel d). In vitro transcribed 16S rRNA (lane 1) and RNA43 (lane 2) served as size markers. The experiment was performed in triplicate and one representative autoradiograph is shown. ( C ) Schematic of the experimental approach to assess the stability of 70S Δ43 ribosomes in vivo as described in A. ( D ) S30 extracts were prepared before (dotted lines) and 120 min after addition of rifampicin (solid lines) to untreated cells (black lines) or 60 min after induction of mazF expression (red lines) and subjected to sucrose density gradient analysis. Peaks representing 30S and 50S subunits and 70S ribosomes are indicated. The peak areas of the 30S and 50S subunits (filled areas) and 70S monosomes (hatched area) that were quantified to determine the subunits/monosome ratios are indicated. ( E ) To monitor MazF-mediated processing of the 16S rRNA, RNA was isolated from the fractions comprising the 70S monosomes. RT-PCR analysis using primers S7/X15 (upper panel), specific for both intact 16S rRNA and 16S Δ43 rRNA, and primers S7/Y12 (lower panel), yielding a product only with uncleaved 16S rRNA. NTC: no template control. Below, the binding sites of the primers are given schematically. ( F ) Ribosome sedimentation profiles of cell extracts 30 min after induction of mazF expression. Total RNA was purified from the indicated fractions (top, 30S, 50S, 70S and polysomes), respectively, and tested for the presence of RNA43 by northern blotting. Total RNA purified from the S30 extract withdrawn 30 min upon induction of mazF expression (lane 1), and in vitro transcribed RNA43 (lane 7) served as controls.

    Article Snippet: RNA sample preparation (total RNA and polysomal RNA) from E. coli strain MC4100 F΄ without or with plasmid pSA1 15 min upon induction of mazF expression was described elsewhere ( ). cDNA libraries were prepared using 50–100 ng of the rRNA-depleted RNA with NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, E7420), following the manufacturer's instructions and sequenced on Illumina HiSequ2000 (read length 100bp).

    Techniques: In Vivo, Expressing, Northern Blot, In Vitro, Autoradiography, Isolation, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Sedimentation, Purification

    Model for the reversible stress adaptation of the translational machinery in E. coli . When E. coli encounters stress (red) the endoribonuclease MazF is activated and removes the 3΄-terminal 43 nts (RNA43) from the 16S rRNA incorporated in active ribosomes (i). The resulting 70S Δ43 ribosomes selectively translate MazF-processed mRNAs to adapt protein synthesis to the adverse conditions (ii). Upon stress relief when canonical mRNAs are transcribed again, the specialized ribosomes become redundant. To regenerate these ribosomes the RNA ligase RtcB re-ligates the 16S Δ43 rRNA present in the stress-ribosomes and RNA43 (iii), thereby restoring the translational proficiency of 70S ribosomes to ensure canonical translation during relaxed conditions (green) (iv).

    Journal: Nucleic Acids Research

    Article Title: The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli

    doi: 10.1093/nar/gkw1018

    Figure Lengend Snippet: Model for the reversible stress adaptation of the translational machinery in E. coli . When E. coli encounters stress (red) the endoribonuclease MazF is activated and removes the 3΄-terminal 43 nts (RNA43) from the 16S rRNA incorporated in active ribosomes (i). The resulting 70S Δ43 ribosomes selectively translate MazF-processed mRNAs to adapt protein synthesis to the adverse conditions (ii). Upon stress relief when canonical mRNAs are transcribed again, the specialized ribosomes become redundant. To regenerate these ribosomes the RNA ligase RtcB re-ligates the 16S Δ43 rRNA present in the stress-ribosomes and RNA43 (iii), thereby restoring the translational proficiency of 70S ribosomes to ensure canonical translation during relaxed conditions (green) (iv).

    Article Snippet: RNA sample preparation (total RNA and polysomal RNA) from E. coli strain MC4100 F΄ without or with plasmid pSA1 15 min upon induction of mazF expression was described elsewhere ( ). cDNA libraries were prepared using 50–100 ng of the rRNA-depleted RNA with NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, E7420), following the manufacturer's instructions and sequenced on Illumina HiSequ2000 (read length 100bp).

    Techniques:

    (A) Transcript abundance of all 940 annotated Wolbachia genes (listed by geneID number) reveals over 95% of Wolbachia transcripts from B . malayi adult male RNA were enriched using the Cappable-seq technique. Each transcript is indicated by in red (the FPKM value in the total RNA) and blue (the FPKM value in capped-RNA sample)(B) A closer view (note difference in y-axis scales between panel A and B) of transcript abundance reveals an enrichment in Wolbachia transcripts in the capped RNA sample.

    Journal: PLoS ONE

    Article Title: Removing the needle from the haystack: Enrichment of Wolbachia endosymbiont transcripts from host nematode RNA by Cappable-seq™

    doi: 10.1371/journal.pone.0173186

    Figure Lengend Snippet: (A) Transcript abundance of all 940 annotated Wolbachia genes (listed by geneID number) reveals over 95% of Wolbachia transcripts from B . malayi adult male RNA were enriched using the Cappable-seq technique. Each transcript is indicated by in red (the FPKM value in the total RNA) and blue (the FPKM value in capped-RNA sample)(B) A closer view (note difference in y-axis scales between panel A and B) of transcript abundance reveals an enrichment in Wolbachia transcripts in the capped RNA sample.

    Article Snippet: Libraries were prepared from capped RNA, uncapped RNA (from bead wash steps above) and total RNA (12.9 ng/1.5 ng, 100 ng/100 ng and 100 ng/70 ng from B . malayi MF/adult males, respectively), using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (New England Biolabs) according to the manufacturer instructions.

    Techniques:

    (A) Transcript abundance of all 940 annotated Wolbachia genes (listed by geneID number) reveals over 95% of Wolbachia transcripts from B . malayi MF RNA were enriched using the Cappable-seq technique. Each transcript is indicated by in red (the FPKM value in the total RNA) and blue (the FPKM value in capped-RNA sample)(B) A closer view (note difference in y-axis scales between panel A and B) of transcript abundance reveals an enrichment in Wolbachia transcripts in the capped RNA sample.

    Journal: PLoS ONE

    Article Title: Removing the needle from the haystack: Enrichment of Wolbachia endosymbiont transcripts from host nematode RNA by Cappable-seq™

    doi: 10.1371/journal.pone.0173186

    Figure Lengend Snippet: (A) Transcript abundance of all 940 annotated Wolbachia genes (listed by geneID number) reveals over 95% of Wolbachia transcripts from B . malayi MF RNA were enriched using the Cappable-seq technique. Each transcript is indicated by in red (the FPKM value in the total RNA) and blue (the FPKM value in capped-RNA sample)(B) A closer view (note difference in y-axis scales between panel A and B) of transcript abundance reveals an enrichment in Wolbachia transcripts in the capped RNA sample.

    Article Snippet: Libraries were prepared from capped RNA, uncapped RNA (from bead wash steps above) and total RNA (12.9 ng/1.5 ng, 100 ng/100 ng and 100 ng/70 ng from B . malayi MF/adult males, respectively), using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (New England Biolabs) according to the manufacturer instructions.

    Techniques:

    A. Transcript coverage (FPKM) of Wolbachia genes reveals over 88% of Wolbachia transcripts from B . malayi adult male RNA were enriched using the Cappable-seq technique. A closer view of transcript abundance (inset) reveals most Wolbachia transcripts in total RNA are present in very low abundance, whereas the Wolbachia transcripts are more abundant in the capped RNA sample. Points along the y-axis are indicative of Wolbachia transcripts that were undetectable in total RNA that were detected in the capped RNA sample. B. Transcript coverage (FPKM) of Wolbachia genes reveals over 95% of Wolbachia transcripts from B . malayi MF RNA were enriched using the Cappable-seq technique. A closer view of transcript abundance (inset) reveals most Wolbachia transcripts in total RNA are present in very low abundance, whereas the Wolbachia transcripts are more abundant in the capped RNA sample. Points along the y-axis are indicative of Wolbachia transcripts that were undetectable in total RNA that were detected in the capped RNA sample.

    Journal: PLoS ONE

    Article Title: Removing the needle from the haystack: Enrichment of Wolbachia endosymbiont transcripts from host nematode RNA by Cappable-seq™

    doi: 10.1371/journal.pone.0173186

    Figure Lengend Snippet: A. Transcript coverage (FPKM) of Wolbachia genes reveals over 88% of Wolbachia transcripts from B . malayi adult male RNA were enriched using the Cappable-seq technique. A closer view of transcript abundance (inset) reveals most Wolbachia transcripts in total RNA are present in very low abundance, whereas the Wolbachia transcripts are more abundant in the capped RNA sample. Points along the y-axis are indicative of Wolbachia transcripts that were undetectable in total RNA that were detected in the capped RNA sample. B. Transcript coverage (FPKM) of Wolbachia genes reveals over 95% of Wolbachia transcripts from B . malayi MF RNA were enriched using the Cappable-seq technique. A closer view of transcript abundance (inset) reveals most Wolbachia transcripts in total RNA are present in very low abundance, whereas the Wolbachia transcripts are more abundant in the capped RNA sample. Points along the y-axis are indicative of Wolbachia transcripts that were undetectable in total RNA that were detected in the capped RNA sample.

    Article Snippet: Libraries were prepared from capped RNA, uncapped RNA (from bead wash steps above) and total RNA (12.9 ng/1.5 ng, 100 ng/100 ng and 100 ng/70 ng from B . malayi MF/adult males, respectively), using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (New England Biolabs) according to the manufacturer instructions.

    Techniques:

    Visualization of infection dynamics in the JW18 cell line. (A) Schematic of Wolbachia detection by RNA Fluorescent In Situ Hybridization (FISH) using a sensitive and specific set of 48 5’-fluorescently-labeled oligos that bind in series to the 23s rRNA of the Wolbachia within a host cell. ( B) Wolbachia -infected JW18 cells labeled by 23s rRNA FISH probe can detect different infection levels in a highly specific manner. Scale bar 5μm. ( C) Wolbachia infection within the JW18 population is steadily maintained at 14% of the total cells in the population. Of the Wolbachia infected cells, the majority (73%) of cells have a low Wolbachia infection (1–10 bacteria per cell), 13.5% contain a medium Wolbachia infection (11–30 bacteria), and 13.5% of the infected JW18 cells have a high infection level ( > 30 bacteria) (n = 793 cells). See S1 – S3 Figs for further characterization of JW18 cells.

    Journal: PLoS Pathogens

    Article Title: Whole genome screen reveals a novel relationship between Wolbachia levels and Drosophila host translation

    doi: 10.1371/journal.ppat.1007445

    Figure Lengend Snippet: Visualization of infection dynamics in the JW18 cell line. (A) Schematic of Wolbachia detection by RNA Fluorescent In Situ Hybridization (FISH) using a sensitive and specific set of 48 5’-fluorescently-labeled oligos that bind in series to the 23s rRNA of the Wolbachia within a host cell. ( B) Wolbachia -infected JW18 cells labeled by 23s rRNA FISH probe can detect different infection levels in a highly specific manner. Scale bar 5μm. ( C) Wolbachia infection within the JW18 population is steadily maintained at 14% of the total cells in the population. Of the Wolbachia infected cells, the majority (73%) of cells have a low Wolbachia infection (1–10 bacteria per cell), 13.5% contain a medium Wolbachia infection (11–30 bacteria), and 13.5% of the infected JW18 cells have a high infection level ( > 30 bacteria) (n = 793 cells). See S1 – S3 Figs for further characterization of JW18 cells.

    Article Snippet: After rRNA depletion libraries were prepared according to manufacturer’s instructions using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England BioLabs, E7420L) and NEBNext Multiplex Oligos for Illumina Index Primers Set I (Illumina, E7335).

    Techniques: Infection, In Situ Hybridization, Fluorescence In Situ Hybridization, Labeling

    Genome-wide screening approach to find novel Wolbachia -host interactions in Wolbachia -infected JW18 Drosophila cells. (A) Schematic of screen layout. Wolbachia -infected JW18 cells are seeded into 384-well plates pre-arrayed with the DRSC version 2.0 whole genome RNAi library designed to include dsRNAs that target the whole Drosophila genome. All plates were screened in triplicate. Cells and dsRNA were incubated for 5 days before processing for an automated high-throughput RNA-FISH assay to detect changes in Wolbachia 23s rRNA levels. ( B) Representative image of Wolbachia detection at 20x with the 23s rRNA Wolbachia FISH probe (magenta) in JW18 cells containing a GFP-Jupiter transgene labeling microtubules (grey). ( C) Negative control dsRNA against LacZ not present in our system. ( D - F ) RNAi control against GFP-Jupiter shows efficient knockdown by 90.2% visually ( D , E ) as well as 97.9% reduction in protein levels by Western blot ( F ). ( G, H) Positive control for increasing Wolbachia levels using efficient RNAi-mediated silencing of host gene RpL40 . ( I) Positive control for decreasing Wolbachia levels through treatment with doxycycline for 5 days. ( J ) Quantification of Wolbachia FISH intensity for controls. ( K) Quantification of Wolbachia level fold-change relative to untreated JW18 cells using DNA qPCR. Note: Scale bars in B-D, G, and I represent 20μm.

    Journal: PLoS Pathogens

    Article Title: Whole genome screen reveals a novel relationship between Wolbachia levels and Drosophila host translation

    doi: 10.1371/journal.ppat.1007445

    Figure Lengend Snippet: Genome-wide screening approach to find novel Wolbachia -host interactions in Wolbachia -infected JW18 Drosophila cells. (A) Schematic of screen layout. Wolbachia -infected JW18 cells are seeded into 384-well plates pre-arrayed with the DRSC version 2.0 whole genome RNAi library designed to include dsRNAs that target the whole Drosophila genome. All plates were screened in triplicate. Cells and dsRNA were incubated for 5 days before processing for an automated high-throughput RNA-FISH assay to detect changes in Wolbachia 23s rRNA levels. ( B) Representative image of Wolbachia detection at 20x with the 23s rRNA Wolbachia FISH probe (magenta) in JW18 cells containing a GFP-Jupiter transgene labeling microtubules (grey). ( C) Negative control dsRNA against LacZ not present in our system. ( D - F ) RNAi control against GFP-Jupiter shows efficient knockdown by 90.2% visually ( D , E ) as well as 97.9% reduction in protein levels by Western blot ( F ). ( G, H) Positive control for increasing Wolbachia levels using efficient RNAi-mediated silencing of host gene RpL40 . ( I) Positive control for decreasing Wolbachia levels through treatment with doxycycline for 5 days. ( J ) Quantification of Wolbachia FISH intensity for controls. ( K) Quantification of Wolbachia level fold-change relative to untreated JW18 cells using DNA qPCR. Note: Scale bars in B-D, G, and I represent 20μm.

    Article Snippet: After rRNA depletion libraries were prepared according to manufacturer’s instructions using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England BioLabs, E7420L) and NEBNext Multiplex Oligos for Illumina Index Primers Set I (Illumina, E7335).

    Techniques: Genome Wide, Infection, Incubation, High Throughput Screening Assay, Fluorescence In Situ Hybridization, Labeling, Negative Control, Western Blot, Positive Control, Real-time Polymerase Chain Reaction

    Knockdown of DLK in differentiated Neuro-2a cells. Neuro-2a cells were infected with an empty lentiviral vector (pLKO.1) or with lentivirus expressing mouse DLK shRNAs (sh73 and sh69). After infection and selection with puromycin, cells were subjected to differentiation for 24 h before being processed for total RNA extraction and whole-cell extracts. a The relative mRNA level of DLK in infected cells was analyzed by quantitative RT-PCR, normalized to three housekeeping genes and calculated with the ΔΔ C T method. The value of DLK mRNA expression in control cells (pLKO.1) was arbitrarily set to 1. Data are the mean ± SEM (error bars) from three independent experiments carried out in triplicate. ****, p

    Journal: Neural Development

    Article Title: Dual leucine zipper kinase regulates expression of axon guidance genes in mouse neuronal cells

    doi: 10.1186/s13064-016-0068-8

    Figure Lengend Snippet: Knockdown of DLK in differentiated Neuro-2a cells. Neuro-2a cells were infected with an empty lentiviral vector (pLKO.1) or with lentivirus expressing mouse DLK shRNAs (sh73 and sh69). After infection and selection with puromycin, cells were subjected to differentiation for 24 h before being processed for total RNA extraction and whole-cell extracts. a The relative mRNA level of DLK in infected cells was analyzed by quantitative RT-PCR, normalized to three housekeeping genes and calculated with the ΔΔ C T method. The value of DLK mRNA expression in control cells (pLKO.1) was arbitrarily set to 1. Data are the mean ± SEM (error bars) from three independent experiments carried out in triplicate. ****, p

    Article Snippet: For all samples, RNA integrity numbers were sufficiently high ( > 9.5) to perform mRNA sequencing. mRNA was purified from 5 μg of total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (#E7490, New England Biolabs, Whitby, Ontario) following manufacturer’s instructions. cDNA libraries were then prepared with 25 ng mRNA of each sample using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (#E7420, New England Biolabs) and barcoded by PCR for subsequent multiplexed sequencing.

    Techniques: Infection, Plasmid Preparation, Expressing, Selection, RNA Extraction, Quantitative RT-PCR

    Validation of RNA-seq data by qRT-PCR and Western blot analyses. a The relative mRNA level of DLK and axon guidance genes in infected cells was analyzed by qRT-PCR, normalized to three housekeeping genes and calculated with the ΔΔ C T method. The value of mRNA expression for each gene in control cells (pLKO.1) was arbitrarily set to 1. Data are the mean ± SEM (error bars) from three independent experiments carried out in triplicate. *, p

    Journal: Neural Development

    Article Title: Dual leucine zipper kinase regulates expression of axon guidance genes in mouse neuronal cells

    doi: 10.1186/s13064-016-0068-8

    Figure Lengend Snippet: Validation of RNA-seq data by qRT-PCR and Western blot analyses. a The relative mRNA level of DLK and axon guidance genes in infected cells was analyzed by qRT-PCR, normalized to three housekeeping genes and calculated with the ΔΔ C T method. The value of mRNA expression for each gene in control cells (pLKO.1) was arbitrarily set to 1. Data are the mean ± SEM (error bars) from three independent experiments carried out in triplicate. *, p

    Article Snippet: For all samples, RNA integrity numbers were sufficiently high ( > 9.5) to perform mRNA sequencing. mRNA was purified from 5 μg of total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (#E7490, New England Biolabs, Whitby, Ontario) following manufacturer’s instructions. cDNA libraries were then prepared with 25 ng mRNA of each sample using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (#E7420, New England Biolabs) and barcoded by PCR for subsequent multiplexed sequencing.

    Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Western Blot, Infection, Expressing

    Antisense transcription detected on the opposite strand of some newly identified RNAs through MiSeq RNA sequencing. RT-PCR was performed on RNA extracted from cells collected at an OD 600nm of 6. Lanes 1 to 5 show IGR_1080208, IGR_1141547, IGR_1141775IGR, IGR_2019414 and 2019159, from left to right after cDNA synthesis (+) or after DNase treatment (−). The gel presented here was cropped for clarity purpose.

    Journal: Scientific Reports

    Article Title: sRNA and cis-antisense sRNA identification in Staphylococcus aureus highlights an unusual sRNA gene cluster with one encoding a secreted peptide

    doi: 10.1038/s41598-017-04786-3

    Figure Lengend Snippet: Antisense transcription detected on the opposite strand of some newly identified RNAs through MiSeq RNA sequencing. RT-PCR was performed on RNA extracted from cells collected at an OD 600nm of 6. Lanes 1 to 5 show IGR_1080208, IGR_1141547, IGR_1141775IGR, IGR_2019414 and 2019159, from left to right after cDNA synthesis (+) or after DNase treatment (−). The gel presented here was cropped for clarity purpose.

    Article Snippet: Stranded cDNA libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs).

    Techniques: RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction

    Characterization of an sRNA transcription hotspot in Staphylococcus aureus Newman. ( A ) RNA mapping profile of IGR_11415444 visualized with Artemis. P1, P2, P3, as2, and as3 correspond to the position of probes and/or primers for northern blot and RT-qPCR experiments. ( B ) Identification of multiple transcripts expressed from the positive strand of the Newman genome. Total RNA were extracted from cells collected at an OD 600nm of 6 and tmRNA was used as an internal loading control. ( C ) Relative expression levels of the IGR_1141544 locus as a function of growth phase. The relative cDNA level was determined using HU as an internal control and OD 600nm of 0.5 as a calibrator. The data shown are the means of three independent experiments. A student t-test was performed to determine differences with condition at OD 600nm of 0.5 (* p

    Journal: Scientific Reports

    Article Title: sRNA and cis-antisense sRNA identification in Staphylococcus aureus highlights an unusual sRNA gene cluster with one encoding a secreted peptide

    doi: 10.1038/s41598-017-04786-3

    Figure Lengend Snippet: Characterization of an sRNA transcription hotspot in Staphylococcus aureus Newman. ( A ) RNA mapping profile of IGR_11415444 visualized with Artemis. P1, P2, P3, as2, and as3 correspond to the position of probes and/or primers for northern blot and RT-qPCR experiments. ( B ) Identification of multiple transcripts expressed from the positive strand of the Newman genome. Total RNA were extracted from cells collected at an OD 600nm of 6 and tmRNA was used as an internal loading control. ( C ) Relative expression levels of the IGR_1141544 locus as a function of growth phase. The relative cDNA level was determined using HU as an internal control and OD 600nm of 0.5 as a calibrator. The data shown are the means of three independent experiments. A student t-test was performed to determine differences with condition at OD 600nm of 0.5 (* p

    Article Snippet: Stranded cDNA libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs).

    Techniques: Northern Blot, Quantitative RT-PCR, Expressing

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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-018-25271-5

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

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

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

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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-018-25271-5

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

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

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

    ALYREF binding sites are enriched on exons of mature mRNAs. ( A ) Detection of ALYREF-RNA complexes. RNase I treated (H: 5 U/ml; L: 0.5 U/ml) and 32 P-labeled RNP complexes were immunoprecipitated with or without the ALYREF antibody from normal and ALYREF knockdown cells. After size-separation using denaturing gel electrophoresis, ALYREF-RNA complexes were transferred to a nitrocellulose membrane. The upper panel shows the autoradiograph of the nitrocellulose membrane. The lower panel shows the western blotting result using the indicated antibodies for input of IP. Red box indicates the region that was extracted for subsequent analyses. ( B ) Correlation of ALYREF iCLIP-seq replicates. Clustered ALYREF binding sites in each gene are plotted for three independent biological replicates (Spearman correlation coefficient, R > 0.90 for all comparisons). ( C ) ALYREF binding sites are enriched at mRNA exon. Left pie chart shows the percentage of RNA-seq reads that uniquely mapped to four human genome regions: mRNA exons, mRNA-introns, lncRNAs or others. Right pie chart shows the percentage of clustered ALYREF binding sites mapped to different human genome regions. ( D ) ALYREF mainly binds mature mRNAs. Percentages of the iCLIP tag in ALYREF binding sites specifically mapped to the exon-exon junction (blue) or exon-intron junction (green).

    Journal: Nucleic Acids Research

    Article Title: ALYREF mainly binds to the 5′ and the 3′ regions of the mRNA in vivo

    doi: 10.1093/nar/gkx597

    Figure Lengend Snippet: ALYREF binding sites are enriched on exons of mature mRNAs. ( A ) Detection of ALYREF-RNA complexes. RNase I treated (H: 5 U/ml; L: 0.5 U/ml) and 32 P-labeled RNP complexes were immunoprecipitated with or without the ALYREF antibody from normal and ALYREF knockdown cells. After size-separation using denaturing gel electrophoresis, ALYREF-RNA complexes were transferred to a nitrocellulose membrane. The upper panel shows the autoradiograph of the nitrocellulose membrane. The lower panel shows the western blotting result using the indicated antibodies for input of IP. Red box indicates the region that was extracted for subsequent analyses. ( B ) Correlation of ALYREF iCLIP-seq replicates. Clustered ALYREF binding sites in each gene are plotted for three independent biological replicates (Spearman correlation coefficient, R > 0.90 for all comparisons). ( C ) ALYREF binding sites are enriched at mRNA exon. Left pie chart shows the percentage of RNA-seq reads that uniquely mapped to four human genome regions: mRNA exons, mRNA-introns, lncRNAs or others. Right pie chart shows the percentage of clustered ALYREF binding sites mapped to different human genome regions. ( D ) ALYREF mainly binds mature mRNAs. Percentages of the iCLIP tag in ALYREF binding sites specifically mapped to the exon-exon junction (blue) or exon-intron junction (green).

    Article Snippet: PolyA RNAs were isolated using NEBNext ploy(A) mRNA magnetic isolation module (NEB), and the libraries were generated using NEBNext Ultra directional RNA library prep kit (NEB).

    Techniques: Binding Assay, Labeling, Immunoprecipitation, Nucleic Acid Electrophoresis, Autoradiography, Western Blot, RNA Sequencing Assay

    Genome-wide effect of ALYREF-binding motifs on nucleocytoplasmic mRNA distribution. ( A ) Northern blots to examine the purities of nuclear and cytoplasmic fractions. The U6 snRNA and tRNA-Lys were used as nuclear and cytoplasmic maker, respectively. ( B ) Screenshots of the UCSC genome browser for RNA-seq of MALAT1. The y-axis displays the RPM. ( C ) Analysis of the correlation of ALYREF binding motifs number and the C/N ratios of intronless mRNAs. Intronless mRNAs were divided into three groups based on occurrences of ALYREF binding motifs per 1000 nt. Based on C/N (RPM) ratio in RNA-seq, intronless mRNAs were further divided into fourtwo subgroups. The percentage of mRNA in each subgroup is shown in colored block. Chi-square tests were used to determine the association between occurrences of ALYREF binding motif and C/N ratio, P

    Journal: Nucleic Acids Research

    Article Title: ALYREF mainly binds to the 5′ and the 3′ regions of the mRNA in vivo

    doi: 10.1093/nar/gkx597

    Figure Lengend Snippet: Genome-wide effect of ALYREF-binding motifs on nucleocytoplasmic mRNA distribution. ( A ) Northern blots to examine the purities of nuclear and cytoplasmic fractions. The U6 snRNA and tRNA-Lys were used as nuclear and cytoplasmic maker, respectively. ( B ) Screenshots of the UCSC genome browser for RNA-seq of MALAT1. The y-axis displays the RPM. ( C ) Analysis of the correlation of ALYREF binding motifs number and the C/N ratios of intronless mRNAs. Intronless mRNAs were divided into three groups based on occurrences of ALYREF binding motifs per 1000 nt. Based on C/N (RPM) ratio in RNA-seq, intronless mRNAs were further divided into fourtwo subgroups. The percentage of mRNA in each subgroup is shown in colored block. Chi-square tests were used to determine the association between occurrences of ALYREF binding motif and C/N ratio, P

    Article Snippet: PolyA RNAs were isolated using NEBNext ploy(A) mRNA magnetic isolation module (NEB), and the libraries were generated using NEBNext Ultra directional RNA library prep kit (NEB).

    Techniques: Genome Wide, Binding Assay, Northern Blot, RNA Sequencing Assay, Blocking Assay

    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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gky1305

    Figure Lengend 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.

    Article Snippet: Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit and NEBNext Multiplex Oligos for Illumina (NEB).

    Techniques: 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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gky1305

    Figure Lengend 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

    Article Snippet: Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit and NEBNext Multiplex Oligos for Illumina (NEB).

    Techniques: Whisker Assay, Labeling

    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

    ( 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

    ERCC reads to determine library preparation quality and back-calculate RNA input mass. (A) After normalizing to RNA input mass, reads aligning to the 92 ERCC spike-in transcripts correlate linearly with ERCC spike-in concentration across six orders of magnitude in all libraries prepared with the miniaturized protocol (R 2 E R C C m a s s ( p g ) T o t a l m a s s ( p g ) = E R C C r e a d s T o t a l r e a d s Back-calculated masses of HeLa libraries correlated strongly with QuBit quantification (R 2 = 0.9954).

    Journal: PLoS ONE

    Article Title: Miniaturization and optimization of 384-well compatible RNA sequencing library preparation

    doi: 10.1371/journal.pone.0206194

    Figure Lengend Snippet: ERCC reads to determine library preparation quality and back-calculate RNA input mass. (A) After normalizing to RNA input mass, reads aligning to the 92 ERCC spike-in transcripts correlate linearly with ERCC spike-in concentration across six orders of magnitude in all libraries prepared with the miniaturized protocol (R 2 E R C C m a s s ( p g ) T o t a l m a s s ( p g ) = E R C C r e a d s T o t a l r e a d s Back-calculated masses of HeLa libraries correlated strongly with QuBit quantification (R 2 = 0.9954).

    Article Snippet: To optimize miniaturization of our laboratory’s current library preparation protocol, we prepared libraries from varying concentrations of HeLa RNA using the New England Biolabs Ultra II RNA Library Prep Kit (E7770S/L).

    Techniques: Concentration Assay

    HeLa transcriptome coverage is comparable in full volume and miniaturized volume preparations. Rank-rank plots of the human transcriptome show strong correlation between the full-volume hand prepared protocol and the miniaturized, automated protocol for both (A) 1ng and (B) 5ng of HeLa RNA input (5ng RNA input: Spearman’s ρ = 0.79, p

    Journal: PLoS ONE

    Article Title: Miniaturization and optimization of 384-well compatible RNA sequencing library preparation

    doi: 10.1371/journal.pone.0206194

    Figure Lengend Snippet: HeLa transcriptome coverage is comparable in full volume and miniaturized volume preparations. Rank-rank plots of the human transcriptome show strong correlation between the full-volume hand prepared protocol and the miniaturized, automated protocol for both (A) 1ng and (B) 5ng of HeLa RNA input (5ng RNA input: Spearman’s ρ = 0.79, p

    Article Snippet: To optimize miniaturization of our laboratory’s current library preparation protocol, we prepared libraries from varying concentrations of HeLa RNA using the New England Biolabs Ultra II RNA Library Prep Kit (E7770S/L).

    Techniques:

    Dehydrated RNA demonstrates preserved integrity. Bioanalyzer traces and RNA Integrity Numbers (RINs) of biological replicates of HeLa RNA. (A) Before being dried in a vacuum evaporator. (B) After being dried for 30 minutes at 40°C. (C) After being dried for 25 minutes at 65°C. RINs indicate that RNA quality is not compromised during the dehydration process.

    Journal: PLoS ONE

    Article Title: Miniaturization and optimization of 384-well compatible RNA sequencing library preparation

    doi: 10.1371/journal.pone.0206194

    Figure Lengend Snippet: Dehydrated RNA demonstrates preserved integrity. Bioanalyzer traces and RNA Integrity Numbers (RINs) of biological replicates of HeLa RNA. (A) Before being dried in a vacuum evaporator. (B) After being dried for 30 minutes at 40°C. (C) After being dried for 25 minutes at 65°C. RINs indicate that RNA quality is not compromised during the dehydration process.

    Article Snippet: To optimize miniaturization of our laboratory’s current library preparation protocol, we prepared libraries from varying concentrations of HeLa RNA using the New England Biolabs Ultra II RNA Library Prep Kit (E7770S/L).

    Techniques:

    Final libraries produced by the full volume and miniaturized protocols have similar fragment distributions. (A) Bioanalyzer trace of a 5ng HeLa RNA final library prepared with the full volume protocol (average fragment size = 438bp, 95% between 200-1000bp). (B) Bioanalyzer trace of a 5ng HeLa RNA final library prepared with the miniaturized protocol (average fragment size = 457bp, 100% between 200-1000bp).

    Journal: PLoS ONE

    Article Title: Miniaturization and optimization of 384-well compatible RNA sequencing library preparation

    doi: 10.1371/journal.pone.0206194

    Figure Lengend Snippet: Final libraries produced by the full volume and miniaturized protocols have similar fragment distributions. (A) Bioanalyzer trace of a 5ng HeLa RNA final library prepared with the full volume protocol (average fragment size = 438bp, 95% between 200-1000bp). (B) Bioanalyzer trace of a 5ng HeLa RNA final library prepared with the miniaturized protocol (average fragment size = 457bp, 100% between 200-1000bp).

    Article Snippet: To optimize miniaturization of our laboratory’s current library preparation protocol, we prepared libraries from varying concentrations of HeLa RNA using the New England Biolabs Ultra II RNA Library Prep Kit (E7770S/L).

    Techniques: Produced