ultra rna library prep kit  (New England Biolabs)


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    NEBNext Ultra RNA Library Prep Kit for Illumina
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    NEBNext Ultra RNA Library Prep Kit for Illumina 96 rxns
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    e7530l
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    mRNA Template Preparation for PCR
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    New England Biolabs ultra rna library prep kit
    NEBNext Ultra RNA Library Prep Kit for Illumina
    NEBNext Ultra RNA Library Prep Kit for Illumina 96 rxns
    https://www.bioz.com/result/ultra rna library prep kit/product/New England Biolabs
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    Images

    1) Product Images from "Transgenerational Effects of Extended Dauer Diapause on Starvation Survival and Gene Expression Plasticity in Caenorhabditis elegans"

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

    Journal: Genetics

    doi: 10.1534/genetics.118.301250

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

    Techniques Used: Expressing, RNA Sequencing Assay

    2) Product Images from "Rare cell detection by single cell RNA sequencing as guided by single molecule RNA FISH"

    Article Title: Rare cell detection by single cell RNA sequencing as guided by single molecule RNA FISH

    Journal: Cell systems

    doi: 10.1016/j.cels.2018.01.014

    Estimates of gene expression heterogeneity in single cell RNA sequencing are highly dependent on transcriptome coverage (A) The Gini coefficient measures a gene’s expression distribution and captures rare cell population heterogeneity. (B) Population structure of SOX10 mRNA levels measured by DropSeq (pink), Fluidigm (blue), and single molecule RNA FISH (smRNA FISH, brown). (C) Gini coefficient for six genes measured by DropSeq (left y-axis) binned by levels of transcriptome coverage as well as Gini coefficients measured by smRNA FISH (right y-axis). (D) Pearson correlation between Gini coefficients measured through DropSeq and smRNA FISH across different levels of transcriptome coverage (# genes detected per cell). Error bars represent ± 1 standard deviation across bootstrap replicates. (E,F) Scatter Plot of the correspondence between Gini coefficients for 26 genes measured by both DropSeq and smRNA FISH. (G) Scatter Plot of the correspondence between Gini coefficients for 26 genes measured by Fluidigm and smRNA FISH. (H) Pearson correlation between Gini coefficient estimates measured by DropSeq and smRNA FISH using different population sizes (# of cells) and levels of transcriptome coverage. Error bars represent ± 1 standard deviation across bootstrap replicates. (I) Pearson correlation between Gini coefficient estimates measured by DropSeq and smRNA FISH after subsampling cells with high transcriptome coverage to different degrees of reads depth. Numbers inside the bars represent the number of reads subsampled. The x-axis represents the average number of genes detected across all cells at a given subsample depth. Error bars represent ± 1 standard deviation across bootstrap replicates.
    Figure Legend Snippet: Estimates of gene expression heterogeneity in single cell RNA sequencing are highly dependent on transcriptome coverage (A) The Gini coefficient measures a gene’s expression distribution and captures rare cell population heterogeneity. (B) Population structure of SOX10 mRNA levels measured by DropSeq (pink), Fluidigm (blue), and single molecule RNA FISH (smRNA FISH, brown). (C) Gini coefficient for six genes measured by DropSeq (left y-axis) binned by levels of transcriptome coverage as well as Gini coefficients measured by smRNA FISH (right y-axis). (D) Pearson correlation between Gini coefficients measured through DropSeq and smRNA FISH across different levels of transcriptome coverage (# genes detected per cell). Error bars represent ± 1 standard deviation across bootstrap replicates. (E,F) Scatter Plot of the correspondence between Gini coefficients for 26 genes measured by both DropSeq and smRNA FISH. (G) Scatter Plot of the correspondence between Gini coefficients for 26 genes measured by Fluidigm and smRNA FISH. (H) Pearson correlation between Gini coefficient estimates measured by DropSeq and smRNA FISH using different population sizes (# of cells) and levels of transcriptome coverage. Error bars represent ± 1 standard deviation across bootstrap replicates. (I) Pearson correlation between Gini coefficient estimates measured by DropSeq and smRNA FISH after subsampling cells with high transcriptome coverage to different degrees of reads depth. Numbers inside the bars represent the number of reads subsampled. The x-axis represents the average number of genes detected across all cells at a given subsample depth. Error bars represent ± 1 standard deviation across bootstrap replicates.

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

    3) Product Images from "Integrated analysis of hepatic mRNA and miRNA profiles identified molecular networks and potential biomarkers of NAFLD"

    Article Title: Integrated analysis of hepatic mRNA and miRNA profiles identified molecular networks and potential biomarkers of NAFLD

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25743-8

    Hierarchical cluster of representative mRNA and miRNA expression across biological replicate samples. ( A ) Heatmap of representative mRNAs. ( B ) Heatmap of representative miRNAs. RNA expression level is represented by colors, with bright blue indicating high values and bright yellow indicating low values.
    Figure Legend Snippet: Hierarchical cluster of representative mRNA and miRNA expression across biological replicate samples. ( A ) Heatmap of representative mRNAs. ( B ) Heatmap of representative miRNAs. RNA expression level is represented by colors, with bright blue indicating high values and bright yellow indicating low values.

    Techniques Used: Expressing, RNA Expression

    4) Product Images from "The primary transcriptome, small RNAs and regulation of antimicrobial resistance in Acinetobacter baumannii ATCC 17978"

    Article Title: The primary transcriptome, small RNAs and regulation of antimicrobial resistance in Acinetobacter baumannii ATCC 17978

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky603

    sRNA in A. baumannii ATCC 17978. ( A ) Normalized, mapped sequence reads from RNA-seq show the expression of sRNAs 17, 37, 75, 76, 77, 84, 99 and 100 (yellow arrows). Curved arrows depict TSS identified in this study and lollipop structures are predicted rho-independent terminators. Northern blotting of selected sRNAs are shown to the right. RNA was isolated from ESP and five μg of total RNA was loaded per lane. The sRNA sizes below the individual blots have been predicted from dRNA-seq data. ( B ) Sequence alignment of Group I and Group III sRNAs created with the Geneious Software (v. 8.1.8); colored bases indicate conservation in at least 50% of aligned sequences (A, red; C, blue; G, yellow; T, green). The riboprobes used in Northern blotting are depicted as black bars atop the sRNA alignments.
    Figure Legend Snippet: sRNA in A. baumannii ATCC 17978. ( A ) Normalized, mapped sequence reads from RNA-seq show the expression of sRNAs 17, 37, 75, 76, 77, 84, 99 and 100 (yellow arrows). Curved arrows depict TSS identified in this study and lollipop structures are predicted rho-independent terminators. Northern blotting of selected sRNAs are shown to the right. RNA was isolated from ESP and five μg of total RNA was loaded per lane. The sRNA sizes below the individual blots have been predicted from dRNA-seq data. ( B ) Sequence alignment of Group I and Group III sRNAs created with the Geneious Software (v. 8.1.8); colored bases indicate conservation in at least 50% of aligned sequences (A, red; C, blue; G, yellow; T, green). The riboprobes used in Northern blotting are depicted as black bars atop the sRNA alignments.

    Techniques Used: Sequencing, RNA Sequencing Assay, Expressing, Northern Blot, Isolation, End-sequence Profiling, Software

    5) Product Images from "Rare cell variability and drug-induced reprogramming as a mode of cancer drug resistance"

    Article Title: Rare cell variability and drug-induced reprogramming as a mode of cancer drug resistance

    Journal: Nature

    doi: 10.1038/nature22794

    Rare cell expression of resistance marker genes is coordinated between genes, leading to cells expressing multiple markers a. AXL vs. VEGFC mRNA in individual WM989-A6 melanoma cells. Dotted lines represent thresholds for high/low. Inset tabulates cells. b. Odds ratio for co-expression. Dark gray boxes indicate zero double-positive cells (1 of n=2 biological replicates). c. Co-stain and sort for EGFR and NGFR into 4 populations: double negative and positive, and EGFR/NGFR positive only, followed by 1μM vemurafenib (2 weeks). Resistant colonies circled in the images (1 of n=2 biological replicates). d. Two cells across multiple rounds of hybridization. RNA FISH signal in white; cell nuclei in blue.
    Figure Legend Snippet: Rare cell expression of resistance marker genes is coordinated between genes, leading to cells expressing multiple markers a. AXL vs. VEGFC mRNA in individual WM989-A6 melanoma cells. Dotted lines represent thresholds for high/low. Inset tabulates cells. b. Odds ratio for co-expression. Dark gray boxes indicate zero double-positive cells (1 of n=2 biological replicates). c. Co-stain and sort for EGFR and NGFR into 4 populations: double negative and positive, and EGFR/NGFR positive only, followed by 1μM vemurafenib (2 weeks). Resistant colonies circled in the images (1 of n=2 biological replicates). d. Two cells across multiple rounds of hybridization. RNA FISH signal in white; cell nuclei in blue.

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

    Iterative RNA FISH enables quantification of genes that are expressed in rare cells and control genes that are expressed throughout a population a. RNA counts are consistent whether a gene is probed on the first cycle of iterative RNA FISH or subsequent cycles. Boxplots summarizing RNA FISH mRNA counts for each gene in the 19 gene panel (shown in Fig. 2a ). We probed each gene from the panel in resistant WM989-A6 cells without performing iterative hybridizations (n=1 with further validation performed on a 5 gene panel; note that we used resistant cells because the generally higher expression levels allowed for more robust comparisons). We then performed iterative RNA FISH with all the probes and compared the total mRNA counts. We took image z-stacks of each sample and captured a total 15–25 cells per sample. Expression levels were similar between the first round of hybridization and all subsequent hybridization cycles. The color of the boxplot indicates the hybridization cycle during which we used each probe. The p-value for differences in RNA counts between the cycles are labeled above each plot. Some variability may be due to sampling with genes that have low and/or highly variable expression, and in these instances, we expect some differences in the two count distributions. There is some loss for some genes in later cycles, but we do not believe that affects our qualitative findings of rare, high-expressing cells. b. Housekeeping genes correlate more with each other than with resistance markers and vice-versa. We performed RNA FISH on 8672 non-drugged cells with probes targeting LOXL2 and AXL (both of which exhibit rare-cell expression) and LMNA and GAPDH, both of which are control genes not associated with resistance (1 of n=2 biological replicates shown). We then performed principal component analysis to determine which genes covary with which other genes. We transformed the vector representing the expression levels of each cell into the space spanned by the first two principal components. Arrows represent transformations of unit vectors of the specified gene into this same space. We observed two rough axes of variability, one corresponding to the GAPDH and LMNA and the other to AXL and LOXL2. Thus, these results show that there is substantial covariation in housekeeping genes and in resistance markers, but that these two axes of variation separate. c. Same plot as in panel b, but with the RNA FISH data shown for WM989-A6 in Figure 2b . d. There are subpopulations of cells that have high expression of multiple resistance marker genes. Histogram of number/fraction of cells that have high expression for a particular number of genes simultaneously, both before, immediately after and then 4 weeks after application of drug (1 of n=2 biological replicates shown). We found that immediately after adding drug, there was a large general decrease in the amount of high-expressing cells, but a few cells remained that expressed several marker genes at once. This suggests, but certainly does not prove, that these multi-expressing cells may be the pre-resistant cells. At best, it establishes that such a correspondence is plausible. e. We used RNA FISH analysis to look (in WM989-A6 cells) at the expression of APCDD1 cells, which was identified as a potential marker of drug-induced reprogramming (as opposed to pre-re- sistance). We measured APCDD1 expression in a total of 61,770 (20,030 in replicate 1 and 41,740 in replicate 2) cells before adding drug and 11,452 (7,138 in replicate 1 and 4,314 cells in replicate 2) cells after cells became stably resistant (n=2 biological replicates shown). Given the number of cells analyzed, we expected that roughly 30 cells in the untreated population would be pre-resistant (assuming conservatively that the frequency of pre-resistance is 1:2000), but despite that, we found essentially no cells with APCDD1 expression levels approaching those of even the median resistant cell. Thus, expression of this gene must have changed upon the pre-resistant cell becoming stably resistant in the presence of drug, as opposed to a selection effect in which high levels of expression in pre-resistance cells become prevalent due to those cells surviving rather than reprogramming.
    Figure Legend Snippet: Iterative RNA FISH enables quantification of genes that are expressed in rare cells and control genes that are expressed throughout a population a. RNA counts are consistent whether a gene is probed on the first cycle of iterative RNA FISH or subsequent cycles. Boxplots summarizing RNA FISH mRNA counts for each gene in the 19 gene panel (shown in Fig. 2a ). We probed each gene from the panel in resistant WM989-A6 cells without performing iterative hybridizations (n=1 with further validation performed on a 5 gene panel; note that we used resistant cells because the generally higher expression levels allowed for more robust comparisons). We then performed iterative RNA FISH with all the probes and compared the total mRNA counts. We took image z-stacks of each sample and captured a total 15–25 cells per sample. Expression levels were similar between the first round of hybridization and all subsequent hybridization cycles. The color of the boxplot indicates the hybridization cycle during which we used each probe. The p-value for differences in RNA counts between the cycles are labeled above each plot. Some variability may be due to sampling with genes that have low and/or highly variable expression, and in these instances, we expect some differences in the two count distributions. There is some loss for some genes in later cycles, but we do not believe that affects our qualitative findings of rare, high-expressing cells. b. Housekeeping genes correlate more with each other than with resistance markers and vice-versa. We performed RNA FISH on 8672 non-drugged cells with probes targeting LOXL2 and AXL (both of which exhibit rare-cell expression) and LMNA and GAPDH, both of which are control genes not associated with resistance (1 of n=2 biological replicates shown). We then performed principal component analysis to determine which genes covary with which other genes. We transformed the vector representing the expression levels of each cell into the space spanned by the first two principal components. Arrows represent transformations of unit vectors of the specified gene into this same space. We observed two rough axes of variability, one corresponding to the GAPDH and LMNA and the other to AXL and LOXL2. Thus, these results show that there is substantial covariation in housekeeping genes and in resistance markers, but that these two axes of variation separate. c. Same plot as in panel b, but with the RNA FISH data shown for WM989-A6 in Figure 2b . d. There are subpopulations of cells that have high expression of multiple resistance marker genes. Histogram of number/fraction of cells that have high expression for a particular number of genes simultaneously, both before, immediately after and then 4 weeks after application of drug (1 of n=2 biological replicates shown). We found that immediately after adding drug, there was a large general decrease in the amount of high-expressing cells, but a few cells remained that expressed several marker genes at once. This suggests, but certainly does not prove, that these multi-expressing cells may be the pre-resistant cells. At best, it establishes that such a correspondence is plausible. e. We used RNA FISH analysis to look (in WM989-A6 cells) at the expression of APCDD1 cells, which was identified as a potential marker of drug-induced reprogramming (as opposed to pre-re- sistance). We measured APCDD1 expression in a total of 61,770 (20,030 in replicate 1 and 41,740 in replicate 2) cells before adding drug and 11,452 (7,138 in replicate 1 and 4,314 cells in replicate 2) cells after cells became stably resistant (n=2 biological replicates shown). Given the number of cells analyzed, we expected that roughly 30 cells in the untreated population would be pre-resistant (assuming conservatively that the frequency of pre-resistance is 1:2000), but despite that, we found essentially no cells with APCDD1 expression levels approaching those of even the median resistant cell. Thus, expression of this gene must have changed upon the pre-resistant cell becoming stably resistant in the presence of drug, as opposed to a selection effect in which high levels of expression in pre-resistance cells become prevalent due to those cells surviving rather than reprogramming.

    Techniques Used: Fluorescence In Situ Hybridization, Expressing, Hybridization, Labeling, Sampling, Transformation Assay, Plasmid Preparation, Marker, Stable Transfection, Selection

    RNA FISH on thousands of melanoma cells reveals rare cells that express high levels of resistance marker genes a. Histograms of transcript abundance for resistance marker genes (top) and nonresistance markers (bottom). The vertical lines represent the threshold for designating cells as either “high” or “low” expressing for a particular gene. The cells labeled by the red carpet below the histogram are high expressing, and the cells labeled by the gray carpet are low expressing. The data set shown contains a total of 8672 cells and is one of two biological replicates. b. In an untreated population of cells, rare cells express resistance marker genes at much higher levels than the population average, sometimes at levels similar to the drug resistant state. Boxplots showing the distribution of mRNA counts per cell for untreated WM989-A6 cells and resistant WM989-A6 cells. The untreated data set is the same data as shown in a. For the resistant WM989-A6 cells, we performed iterative RNA FISH with the same panel of genes. The untreated data set contains a total of 8672 cells and the resistant data set contains a total of 4082 cells (1 of n=2 biological replicates are shown for each dataset). Asterisks next to the gene names indicates that the max expression of the untreated sample is greater than or equal to the median of the resistant sample, demonstrating that for these 7 of 9 genes, the “high” cells have expression levels potentially equivalent to resistant cells. However, we also point out that given that the sampling of high expressing cells in the untreated samples is low, it is difficult to explicitly compare the distributions to say that the expression in the rare high-expressing cells is equivalent to that in stably resistant cells. c. Rare cells expressing sporadic but high levels of resistance markers are still present when each gene is normalized by GAPDH mRNA counts. Each histogram shows the distribution of GAPDH normalized counts for a particular jackpot gene. The counts for each gene in each cells has been divided by the GAPDH counts in that same cell. This accounts for any volume-dependent differences between cells. Cells that had GAPDH counts less than 50 were dropped from this analysis (these cells were infrequent and gave abnormally high numbers after normalization, thus were dropped). With these cells removed, the data set contains a total of 8477 cells. d. Heatmap shows the odds ratio for co-expression between all pairs of genes in WM989-A6 cells (1 of n=2 biological replicates shown). Dark gray boxes label pairs where there were zero cells with counts high expression threshold for both genes. The heatmap in the middle has the same thresholds for designating cells as “high” or “low” as used in Fig. 3b . Meanwhile, the heatmap on the left shows the same analysis with the thresholds set to 1⁄2 of the their value in 3b and the heatmap on the right shows this analysis with thresholds set to twice their value in Fig. 3b . When the thresholds are at 1⁄2, the result is very similar to that in Fig. 3b . However, increasing the threshold by 2X leads to many gene pairs that do not have any cells that are “high” for both genes (indicated by the dark gray boxes). e. Heatmap showing odds ratios for WM989-A6 data after 4 weeks in drug (1 of n=2 biological replicates shown).
    Figure Legend Snippet: RNA FISH on thousands of melanoma cells reveals rare cells that express high levels of resistance marker genes a. Histograms of transcript abundance for resistance marker genes (top) and nonresistance markers (bottom). The vertical lines represent the threshold for designating cells as either “high” or “low” expressing for a particular gene. The cells labeled by the red carpet below the histogram are high expressing, and the cells labeled by the gray carpet are low expressing. The data set shown contains a total of 8672 cells and is one of two biological replicates. b. In an untreated population of cells, rare cells express resistance marker genes at much higher levels than the population average, sometimes at levels similar to the drug resistant state. Boxplots showing the distribution of mRNA counts per cell for untreated WM989-A6 cells and resistant WM989-A6 cells. The untreated data set is the same data as shown in a. For the resistant WM989-A6 cells, we performed iterative RNA FISH with the same panel of genes. The untreated data set contains a total of 8672 cells and the resistant data set contains a total of 4082 cells (1 of n=2 biological replicates are shown for each dataset). Asterisks next to the gene names indicates that the max expression of the untreated sample is greater than or equal to the median of the resistant sample, demonstrating that for these 7 of 9 genes, the “high” cells have expression levels potentially equivalent to resistant cells. However, we also point out that given that the sampling of high expressing cells in the untreated samples is low, it is difficult to explicitly compare the distributions to say that the expression in the rare high-expressing cells is equivalent to that in stably resistant cells. c. Rare cells expressing sporadic but high levels of resistance markers are still present when each gene is normalized by GAPDH mRNA counts. Each histogram shows the distribution of GAPDH normalized counts for a particular jackpot gene. The counts for each gene in each cells has been divided by the GAPDH counts in that same cell. This accounts for any volume-dependent differences between cells. Cells that had GAPDH counts less than 50 were dropped from this analysis (these cells were infrequent and gave abnormally high numbers after normalization, thus were dropped). With these cells removed, the data set contains a total of 8477 cells. d. Heatmap shows the odds ratio for co-expression between all pairs of genes in WM989-A6 cells (1 of n=2 biological replicates shown). Dark gray boxes label pairs where there were zero cells with counts high expression threshold for both genes. The heatmap in the middle has the same thresholds for designating cells as “high” or “low” as used in Fig. 3b . Meanwhile, the heatmap on the left shows the same analysis with the thresholds set to 1⁄2 of the their value in 3b and the heatmap on the right shows this analysis with thresholds set to twice their value in Fig. 3b . When the thresholds are at 1⁄2, the result is very similar to that in Fig. 3b . However, increasing the threshold by 2X leads to many gene pairs that do not have any cells that are “high” for both genes (indicated by the dark gray boxes). e. Heatmap showing odds ratios for WM989-A6 data after 4 weeks in drug (1 of n=2 biological replicates shown).

    Techniques Used: Fluorescence In Situ Hybridization, Marker, Expressing, Labeling, Sampling, Stable Transfection

    Resistance to vemurafenib is not heritable, and pre-existing pre-resistant cells are marked by very high expression of resistance genes a. Alternative models for heritability of the resistant phenotype and simulated outcomes of each model. b. Distributions of resistant colonies in WM989-A6 (n=2 biological replicates of 43 and 29 clones; WM983B-E9 in Extended Data Fig. 3 ). c. Transcriptome analysis before drug, 48 hours after drug and stably resistant cultures (see Extended Data Fig. 2 ). Heatmap depicts “marker genes” whose expression increased in resistant cells relative to untreated. d. Computational representation of single-cell RNA FISH (8672 untreated cells) for AXL , NGFR , and EGFR mRNA; each dot is a cell colored by number of mRNA (1 of n=2 biological replicates). e. Single-cell AXL RNA FISH (1966 cells) after 4 weeks treatment with 1μM vemurafenib (1 of n=2 biological replicates). f. FACS of cells with an EGFR antibody; isolated an EGFR-high and mixed cell population, then applied vemurafenib. Two-well chamber of populations after 3 weeks vemurafenib(1 of n=3 biological replicates, Extended Data Fig. 5a ). g. Ratio of number of colonies in EGFR-high vs. mixed wells after cells grew without drug for varying periods before vemurafenib application. Error bars represent standard error of the mean (3 biological replicates).
    Figure Legend Snippet: Resistance to vemurafenib is not heritable, and pre-existing pre-resistant cells are marked by very high expression of resistance genes a. Alternative models for heritability of the resistant phenotype and simulated outcomes of each model. b. Distributions of resistant colonies in WM989-A6 (n=2 biological replicates of 43 and 29 clones; WM983B-E9 in Extended Data Fig. 3 ). c. Transcriptome analysis before drug, 48 hours after drug and stably resistant cultures (see Extended Data Fig. 2 ). Heatmap depicts “marker genes” whose expression increased in resistant cells relative to untreated. d. Computational representation of single-cell RNA FISH (8672 untreated cells) for AXL , NGFR , and EGFR mRNA; each dot is a cell colored by number of mRNA (1 of n=2 biological replicates). e. Single-cell AXL RNA FISH (1966 cells) after 4 weeks treatment with 1μM vemurafenib (1 of n=2 biological replicates). f. FACS of cells with an EGFR antibody; isolated an EGFR-high and mixed cell population, then applied vemurafenib. Two-well chamber of populations after 3 weeks vemurafenib(1 of n=3 biological replicates, Extended Data Fig. 5a ). g. Ratio of number of colonies in EGFR-high vs. mixed wells after cells grew without drug for varying periods before vemurafenib application. Error bars represent standard error of the mean (3 biological replicates).

    Techniques Used: Expressing, Clone Assay, Stable Transfection, Fluorescence In Situ Hybridization, FACS, Isolation

    Sorting for EGFR-high cells enriches for pre-resistant cells and removing drug from resistant cells does not appear to reverse the resistant phenotype a. Quantification of 3 biological replicates of the experiment depicted in Fig. 1f . b,c. Histograms showing the transcript abundance measured by RNA FISH in untreated and FACS sorted EGFR-high and mixed cell populations (n=1). The green histograms are from the EGFR-high population and the gray histograms are the mixed population. The percentage of high-expressing cells are labeled on each plot. Panel b shows resistance marker genes EGFR, WNT5A, SERPINE1, and PDGFRβ, and panel c shows melanocyte development genes, SOX10 and MITF, and a housekeeping gene, GAPDH. d. Histograms of percentage of cells that have high expression of a particular number of genes simultaneously. The left histogram is from the FACS sorted EGFR-high cells, and the right histogram is from the mixed population. e. Boxplots summarize the single-cell RNA FISH counts for EGFR and NGFR in flow sorted populations shown in Fig. 3c . These results show that sorting the high populations indeed enriched for EGFR and NGFR mRNA, thus validating the sort procedure (n=1). Furthermore, it shows that the double sorting does not further enrich for either EGFR or NGFR mRNA alone, showing that the effects of the double sort do not arise from a further enrichment of either EGFR or NGFR-high cells per se, but rather the combination of both in the same cell. f. Isolated resistant subclones are stably resistant to vemurafenib. We established stably resistant subclones of WM989-A6 cells grown in vemurafenib by culturing genetically homogeneous WM989-A6 subclones, adding drug, then isolating small resistant colonies and expanding them in the presence of drug into large populations. For three such resistant subclones, we removed drug for a period of three weeks (drug “holiday”), then added drug back for a week and looked for response. Generally, the cells looked fairly similar to the pre-holiday state and continued to proliferate, indicating that they remained insensitive to drug despite the prolonged holiday from drug exposure. The bottom panel is a control experiment consisting of a non-resistant parental line exposed to drug, showing the morphological changes associated with drug response.
    Figure Legend Snippet: Sorting for EGFR-high cells enriches for pre-resistant cells and removing drug from resistant cells does not appear to reverse the resistant phenotype a. Quantification of 3 biological replicates of the experiment depicted in Fig. 1f . b,c. Histograms showing the transcript abundance measured by RNA FISH in untreated and FACS sorted EGFR-high and mixed cell populations (n=1). The green histograms are from the EGFR-high population and the gray histograms are the mixed population. The percentage of high-expressing cells are labeled on each plot. Panel b shows resistance marker genes EGFR, WNT5A, SERPINE1, and PDGFRβ, and panel c shows melanocyte development genes, SOX10 and MITF, and a housekeeping gene, GAPDH. d. Histograms of percentage of cells that have high expression of a particular number of genes simultaneously. The left histogram is from the FACS sorted EGFR-high cells, and the right histogram is from the mixed population. e. Boxplots summarize the single-cell RNA FISH counts for EGFR and NGFR in flow sorted populations shown in Fig. 3c . These results show that sorting the high populations indeed enriched for EGFR and NGFR mRNA, thus validating the sort procedure (n=1). Furthermore, it shows that the double sorting does not further enrich for either EGFR or NGFR mRNA alone, showing that the effects of the double sort do not arise from a further enrichment of either EGFR or NGFR-high cells per se, but rather the combination of both in the same cell. f. Isolated resistant subclones are stably resistant to vemurafenib. We established stably resistant subclones of WM989-A6 cells grown in vemurafenib by culturing genetically homogeneous WM989-A6 subclones, adding drug, then isolating small resistant colonies and expanding them in the presence of drug into large populations. For three such resistant subclones, we removed drug for a period of three weeks (drug “holiday”), then added drug back for a week and looked for response. Generally, the cells looked fairly similar to the pre-holiday state and continued to proliferate, indicating that they remained insensitive to drug despite the prolonged holiday from drug exposure. The bottom panel is a control experiment consisting of a non-resistant parental line exposed to drug, showing the morphological changes associated with drug response.

    Techniques Used: Fluorescence In Situ Hybridization, FACS, Expressing, Labeling, Marker, Flow Cytometry, Isolation, Stable Transfection

    Analysis of multiple patient-derived xenografts reveals cells that sporadically express high levels of some resistance markers a. Table summarizing results of our patient-derived xenograft experiments, including the 4 different models and all the genes tested with each. b. Histograms show full distribution of mRNA expression for genes for which we saw convincing signal. Note that for some expressing genes, there were sporadic noise spots in the analysis, leading to some cells with, say, transcript counts of 1–2 that are probably spurious. c. Image panel of marker gene expression in the patient-derived xenografts. d. Computational representation of CYR61 mRNA expression in patient-derived xenografts. Each cell is represented by a dot on this plot and the color of the dot represents the number of RNA in that particular cell as indicated by the color scale bar. e. Histograms show full distribution of mRNA expression for CYR61 and LOXL2 in WM4335.
    Figure Legend Snippet: Analysis of multiple patient-derived xenografts reveals cells that sporadically express high levels of some resistance markers a. Table summarizing results of our patient-derived xenograft experiments, including the 4 different models and all the genes tested with each. b. Histograms show full distribution of mRNA expression for genes for which we saw convincing signal. Note that for some expressing genes, there were sporadic noise spots in the analysis, leading to some cells with, say, transcript counts of 1–2 that are probably spurious. c. Image panel of marker gene expression in the patient-derived xenografts. d. Computational representation of CYR61 mRNA expression in patient-derived xenografts. Each cell is represented by a dot on this plot and the color of the dot represents the number of RNA in that particular cell as indicated by the color scale bar. e. Histograms show full distribution of mRNA expression for CYR61 and LOXL2 in WM4335.

    Techniques Used: Derivative Assay, Expressing, Marker

    6) Product Images from "Enhanced Anti-lymphoma Activity of CAR19-iNKT Cells Underpinned by Dual CD19 and CD1d Targeting"

    Article Title: Enhanced Anti-lymphoma Activity of CAR19-iNKT Cells Underpinned by Dual CD19 and CD1d Targeting

    Journal: Cancer Cell

    doi: 10.1016/j.ccell.2018.08.017

    Transcriptional and Epigenetic Basis for Enhancing CAR19-iNKT Cell Reactivity (A) CD1D mRNA quantification by qPCR in CLL cells from two patients upon ATRA treatment (10 −6 M) for 0–96 hr. (B and C) Bar charts (B) and flow cytometry histograms (C) showing CD1d expression on malignant B cells upon ATRA treatment and mean fluorescent intensity (MFI) analysis of CD1d expression in comparison with isotype control. (D) Cytotoxicity of second- and third-generation CAR19-T and -NKT cells against αGalCer-pulsed CLL cells pre-treated with 0.1% DMSO control or 10 −6 M ATRA. Error bars represent SEM of triplicate assays. (E) ChIP-qPCR assay for H3K4me3 and H3K27me3 enrichment in the promoter of CD1D using IgG as control in U266 cells. GAPDH is an active gene control, while HOXA2 is a repressed gene control. ChIP data are shown as a percentage of the input chromatin. (F) ChIP-re-ChIP qPCR assay showing fold enrichment of H3K27me3 or IgG control after immunoprecipitation (IP) against H3K4me3. (G) ChIP-qPCR assay against RNA Pol II for Ser5 over Ser2 phosphorylated form at the promoter of the indicated genes. (H) ChIP-qPCR assay against RARα, EZH2, and Ig control at the promoters of the genes shown. (I) ChIP-re-ChIP qPCR assay showing enrichment of EZH2 or IgG control after IP against RARα in U266 cells for –(I) (n = 3). (J) qPCR quantification of CD1D mRNA in U266 cells treated with 0.1% DMSO, 10 −6 M GSK343, 10 −6 M ATRA or 10 −6 M GSK343 plus 10 −6 M ATRA. Values are normalized to CD1D mRNA expression levels in normal peripheral PB B cells (n = 3). ND, not detectable. (K and L) Relative MFI analysis (K) and histogram depiction (L) of CD1d expression in comparison with isotype control in U266 cells from the same experiment shown in (J). Error bars represent SEM. See also Figure S4 .
    Figure Legend Snippet: Transcriptional and Epigenetic Basis for Enhancing CAR19-iNKT Cell Reactivity (A) CD1D mRNA quantification by qPCR in CLL cells from two patients upon ATRA treatment (10 −6 M) for 0–96 hr. (B and C) Bar charts (B) and flow cytometry histograms (C) showing CD1d expression on malignant B cells upon ATRA treatment and mean fluorescent intensity (MFI) analysis of CD1d expression in comparison with isotype control. (D) Cytotoxicity of second- and third-generation CAR19-T and -NKT cells against αGalCer-pulsed CLL cells pre-treated with 0.1% DMSO control or 10 −6 M ATRA. Error bars represent SEM of triplicate assays. (E) ChIP-qPCR assay for H3K4me3 and H3K27me3 enrichment in the promoter of CD1D using IgG as control in U266 cells. GAPDH is an active gene control, while HOXA2 is a repressed gene control. ChIP data are shown as a percentage of the input chromatin. (F) ChIP-re-ChIP qPCR assay showing fold enrichment of H3K27me3 or IgG control after immunoprecipitation (IP) against H3K4me3. (G) ChIP-qPCR assay against RNA Pol II for Ser5 over Ser2 phosphorylated form at the promoter of the indicated genes. (H) ChIP-qPCR assay against RARα, EZH2, and Ig control at the promoters of the genes shown. (I) ChIP-re-ChIP qPCR assay showing enrichment of EZH2 or IgG control after IP against RARα in U266 cells for –(I) (n = 3). (J) qPCR quantification of CD1D mRNA in U266 cells treated with 0.1% DMSO, 10 −6 M GSK343, 10 −6 M ATRA or 10 −6 M GSK343 plus 10 −6 M ATRA. Values are normalized to CD1D mRNA expression levels in normal peripheral PB B cells (n = 3). ND, not detectable. (K and L) Relative MFI analysis (K) and histogram depiction (L) of CD1d expression in comparison with isotype control in U266 cells from the same experiment shown in (J). Error bars represent SEM. See also Figure S4 .

    Techniques Used: Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Expressing, Chromatin Immunoprecipitation, Immunoprecipitation

    7) Product Images from "Integrated analysis of hepatic mRNA and miRNA profiles identified molecular networks and potential biomarkers of NAFLD"

    Article Title: Integrated analysis of hepatic mRNA and miRNA profiles identified molecular networks and potential biomarkers of NAFLD

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25743-8

    Real-time PCR validation of several representative expressed mRNAs and miRNAs. The x-axis represents RNA names, and the y-axis represents log 2 (fold change) based on the ratios between the NAFLD and normal groups’ average expression values. Blue bars represent data yielded by real-time qPCR, and red points represent data obtained by RNA sequencing.
    Figure Legend Snippet: Real-time PCR validation of several representative expressed mRNAs and miRNAs. The x-axis represents RNA names, and the y-axis represents log 2 (fold change) based on the ratios between the NAFLD and normal groups’ average expression values. Blue bars represent data yielded by real-time qPCR, and red points represent data obtained by RNA sequencing.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, RNA Sequencing Assay

    Hierarchical cluster of representative mRNA and miRNA expression across biological replicate samples. ( A ) Heatmap of representative mRNAs. ( B ) Heatmap of representative miRNAs. RNA expression level is represented by colors, with bright blue indicating high values and bright yellow indicating low values.
    Figure Legend Snippet: Hierarchical cluster of representative mRNA and miRNA expression across biological replicate samples. ( A ) Heatmap of representative mRNAs. ( B ) Heatmap of representative miRNAs. RNA expression level is represented by colors, with bright blue indicating high values and bright yellow indicating low values.

    Techniques Used: Expressing, RNA Expression

    8) Product Images from "RNA N6-methyladenosine demethylase FTO promotes breast tumor progression through inhibiting BNIP3"

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

    Journal: Molecular Cancer

    doi: 10.1186/s12943-019-1004-4

    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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

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

    9) Product Images from "Effects of anticholinergic agent on miRNA profiles and transcriptomes in a murine model of allergic rhinitis"

    Article Title: Effects of anticholinergic agent on miRNA profiles and transcriptomes in a murine model of allergic rhinitis

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2017.7411

    Venn diagram of differentially expressed (A) mRNAs and (B) miRNAs. Control: Group A (n=7) and group B (n=8). Model: Group C (n=8), group D (n=10) and group E (n=9). When mRNAs or miRNAs exhibit similar characteristics, they appear in the overlapping boxes. mRNA, messenger RNA; miRNA, microRNA.
    Figure Legend Snippet: Venn diagram of differentially expressed (A) mRNAs and (B) miRNAs. Control: Group A (n=7) and group B (n=8). Model: Group C (n=8), group D (n=10) and group E (n=9). When mRNAs or miRNAs exhibit similar characteristics, they appear in the overlapping boxes. mRNA, messenger RNA; miRNA, microRNA.

    Techniques Used:

    10) Product Images from "Propranolol exhibits activity against hemangiomas independent of beta blockade"

    Article Title: Propranolol exhibits activity against hemangiomas independent of beta blockade

    Journal: NPJ Precision Oncology

    doi: 10.1038/s41698-019-0099-9

    Heat map of differentially expressed RNA between bEnd.3 cells treated with R-propranolol and vehicle. Twenty-four transcripts were identified by RNAseq to be upregulated or downregulated in R-propranolol-treated cells when compared to the vehicle-treated control cells with p value ≤ 0.05. Transcripts were further filtered by minimum two-fold difference in the expression levels
    Figure Legend Snippet: Heat map of differentially expressed RNA between bEnd.3 cells treated with R-propranolol and vehicle. Twenty-four transcripts were identified by RNAseq to be upregulated or downregulated in R-propranolol-treated cells when compared to the vehicle-treated control cells with p value ≤ 0.05. Transcripts were further filtered by minimum two-fold difference in the expression levels

    Techniques Used: Expressing

    11) Product Images from "Transcriptomic Analysis of Differentially Expressed Genes during Flower Organ Development in Genetic Male Sterile and Male Fertile Tagetes erecta by Digital Gene-Expression Profiling"

    Article Title: Transcriptomic Analysis of Differentially Expressed Genes during Flower Organ Development in Genetic Male Sterile and Male Fertile Tagetes erecta by Digital Gene-Expression Profiling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0150892

    Linear regression analysis of the fold change of the gene expression ratios between DEG sequencing and qRT-PCR. 26 unigenes were selected for quantitative real-time PCR analysis to confirm the accuracy and reproducibility of the Illumina expression profiles using the same RNA samples that were used for DGE sequencing. The relative expression levels of the genes were calculated using the 2 −ΔΔCt method in qRT-PCR analysis. The DGE sequencing data were represented by the FPKM value of samples. Scatterplots were generated by the log 2 expression ratios from DGE sequencing data (x-axis) and qRT-PCR data (y-axis).
    Figure Legend Snippet: Linear regression analysis of the fold change of the gene expression ratios between DEG sequencing and qRT-PCR. 26 unigenes were selected for quantitative real-time PCR analysis to confirm the accuracy and reproducibility of the Illumina expression profiles using the same RNA samples that were used for DGE sequencing. The relative expression levels of the genes were calculated using the 2 −ΔΔCt method in qRT-PCR analysis. The DGE sequencing data were represented by the FPKM value of samples. Scatterplots were generated by the log 2 expression ratios from DGE sequencing data (x-axis) and qRT-PCR data (y-axis).

    Techniques Used: Expressing, Sequencing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Generated

    12) Product Images from "SIRT7-dependent deacetylation of the U3-55k protein controls pre-rRNA processing"

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

    Journal: Nature Communications

    doi: 10.1038/ncomms10734

    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
    Figure Legend 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

    Techniques Used: 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

    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
    Figure Legend 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

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

    13) Product Images from "Finding Nemo: hybrid assembly with Oxford Nanopore and Illumina reads greatly improves the clownfish (Amphiprion ocellaris) genome assembly"

    Article Title: Finding Nemo: hybrid assembly with Oxford Nanopore and Illumina reads greatly improves the clownfish (Amphiprion ocellaris) genome assembly

    Journal: GigaScience

    doi: 10.1093/gigascience/gix137

    Mapping of MinION long reads, Illumina-assembled scaffolds, and RNA-sequencing reads of male and female A. ocellaris to the genomic region containing the cyp19a1a gene. Transcripts per million (TPM) values were calculated using Kallisto, version 0.43.1 [ 46 ].
    Figure Legend Snippet: Mapping of MinION long reads, Illumina-assembled scaffolds, and RNA-sequencing reads of male and female A. ocellaris to the genomic region containing the cyp19a1a gene. Transcripts per million (TPM) values were calculated using Kallisto, version 0.43.1 [ 46 ].

    Techniques Used: RNA Sequencing Assay

    14) Product Images from "In vivo RNA editing of point mutations via RNA-guided adenosine deaminases"

    Article Title: In vivo RNA editing of point mutations via RNA-guided adenosine deaminases

    Journal: Nature methods

    doi: 10.1038/s41592-019-0323-0

    In vivo RNA editing in mouse models of human disease: (a) Schematic of the DNA and RNA targeting approaches to restore dystrophin expression in the mdx mouse model of Duchenne Muscular Dystrophy: (i) a dual gRNA-CRISPR based approach leading to in frame excision of exon 23 and (ii) ADAR2 and MCP-ADAR1 based editing of the ochre codon. (b) Immunofluorescence staining for dystrophin in the TA muscle shows partial restoration of expression in treated samples (intra-muscular injections of AAV8-ADAR2, AAV8-ADAR2 (E488Q), and AAV8-CRISPR). Partial restoration of nNOS localization is also seen in treated samples (scale bar: 250μm). (c) In vivo TAA- > TGG/TAG/TGA RNA editing efficiencies in corresponding treated adult mdx mice. Values represent mean +/− SEM (n=4, 3, 7, 3, 3, 10, 3, 4 independent TA muscles respectively). (d) Schematic of the OTC locus in the spf ash mouse model of Ornithine Transcarbamylase deficiency which have a G- > A point mutation at a donor splice site in the last nucleotide of exon 4, and approach for correction of mutant OTC mRNA via ADAR2 mediated RNA editing. (e) In vivo RNA correction efficiencies in the correctly spliced OTC mRNA in the livers of treated adult spf ash mice (retro-orbital injections of AAV8-ADAR2 and AAV8-ADAR2 (E488Q)). Values represent mean +/− SEM (n=4, 4, 3, 3, 4, 5 independent animals respectively).
    Figure Legend Snippet: In vivo RNA editing in mouse models of human disease: (a) Schematic of the DNA and RNA targeting approaches to restore dystrophin expression in the mdx mouse model of Duchenne Muscular Dystrophy: (i) a dual gRNA-CRISPR based approach leading to in frame excision of exon 23 and (ii) ADAR2 and MCP-ADAR1 based editing of the ochre codon. (b) Immunofluorescence staining for dystrophin in the TA muscle shows partial restoration of expression in treated samples (intra-muscular injections of AAV8-ADAR2, AAV8-ADAR2 (E488Q), and AAV8-CRISPR). Partial restoration of nNOS localization is also seen in treated samples (scale bar: 250μm). (c) In vivo TAA- > TGG/TAG/TGA RNA editing efficiencies in corresponding treated adult mdx mice. Values represent mean +/− SEM (n=4, 3, 7, 3, 3, 10, 3, 4 independent TA muscles respectively). (d) Schematic of the OTC locus in the spf ash mouse model of Ornithine Transcarbamylase deficiency which have a G- > A point mutation at a donor splice site in the last nucleotide of exon 4, and approach for correction of mutant OTC mRNA via ADAR2 mediated RNA editing. (e) In vivo RNA correction efficiencies in the correctly spliced OTC mRNA in the livers of treated adult spf ash mice (retro-orbital injections of AAV8-ADAR2 and AAV8-ADAR2 (E488Q)). Values represent mean +/− SEM (n=4, 4, 3, 3, 4, 5 independent animals respectively).

    Techniques Used: In Vivo, Expressing, CRISPR, Immunofluorescence, Staining, Mouse Assay, Mutagenesis

    15) Product Images from "SDG8-Mediated Histone Methylation and RNA Processing Function in the Response to Nitrate Signaling"

    Article Title: SDG8-Mediated Histone Methylation and RNA Processing Function in the Response to Nitrate Signaling

    Journal: Plant Physiology

    doi: 10.1104/pp.19.00682

    A putative transporter displays RNA isoform switching in response to N. A, Expression profile of AT3G54830 (encoding a putative transmembrane transporter) shows a distinct preference for RNA isoforms under the N-supplied condition (+N, green, long RNA isoform) compared to the N-starved condition (−N, blue, short RNA isoform). Specifically, a gene-centric view of RNA-Seq sequencing depth is shown with three biological replicates for each condition. B, Predicted structure of proteins encoded by the long or short mRNA isoform suggested that the encoded protein is likely a putative transmembrane transporter, with distinct structures under N-supplied versus N-starved conditions. Structures were predicted using the program Phyre2.
    Figure Legend Snippet: A putative transporter displays RNA isoform switching in response to N. A, Expression profile of AT3G54830 (encoding a putative transmembrane transporter) shows a distinct preference for RNA isoforms under the N-supplied condition (+N, green, long RNA isoform) compared to the N-starved condition (−N, blue, short RNA isoform). Specifically, a gene-centric view of RNA-Seq sequencing depth is shown with three biological replicates for each condition. B, Predicted structure of proteins encoded by the long or short mRNA isoform suggested that the encoded protein is likely a putative transmembrane transporter, with distinct structures under N-supplied versus N-starved conditions. Structures were predicted using the program Phyre2.

    Techniques Used: Expressing, RNA Sequencing Assay, Sequencing

    16) Product Images from "The primary transcriptome, small RNAs and regulation of antimicrobial resistance in Acinetobacter baumannii ATCC 17978"

    Article Title: The primary transcriptome, small RNAs and regulation of antimicrobial resistance in Acinetobacter baumannii ATCC 17978

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky603

    sRNA in A. baumannii ATCC 17978. ( A ) Normalized, mapped sequence reads from RNA-seq show the expression of sRNAs 17, 37, 75, 76, 77, 84, 99 and 100 (yellow arrows). Curved arrows depict TSS identified in this study and lollipop structures are predicted rho-independent terminators. Northern blotting of selected sRNAs are shown to the right. RNA was isolated from ESP and five μg of total RNA was loaded per lane. The sRNA sizes below the individual blots have been predicted from dRNA-seq data. ( B ) Sequence alignment of Group I and Group III sRNAs created with the Geneious Software (v. 8.1.8); colored bases indicate conservation in at least 50% of aligned sequences (A, red; C, blue; G, yellow; T, green). The riboprobes used in Northern blotting are depicted as black bars atop the sRNA alignments.
    Figure Legend Snippet: sRNA in A. baumannii ATCC 17978. ( A ) Normalized, mapped sequence reads from RNA-seq show the expression of sRNAs 17, 37, 75, 76, 77, 84, 99 and 100 (yellow arrows). Curved arrows depict TSS identified in this study and lollipop structures are predicted rho-independent terminators. Northern blotting of selected sRNAs are shown to the right. RNA was isolated from ESP and five μg of total RNA was loaded per lane. The sRNA sizes below the individual blots have been predicted from dRNA-seq data. ( B ) Sequence alignment of Group I and Group III sRNAs created with the Geneious Software (v. 8.1.8); colored bases indicate conservation in at least 50% of aligned sequences (A, red; C, blue; G, yellow; T, green). The riboprobes used in Northern blotting are depicted as black bars atop the sRNA alignments.

    Techniques Used: Sequencing, RNA Sequencing Assay, Expressing, Northern Blot, Isolation, End-sequence Profiling, Software

    17) Product Images from "Multivalent binding of PWWP2A to H2A.Z regulates mitosis and neural crest differentiation"

    Article Title: Multivalent binding of PWWP2A to H2A.Z regulates mitosis and neural crest differentiation

    Journal: The EMBO Journal

    doi: 10.15252/embj.201695757

    Analysis of Xenopus pwwp2a expression and function Expression pattern of endogenous Xenopus tropicalis pwwp2a mRNA during early development. Pictures show whole‐mount RNA in situ hybridization patterns (purple color) for the following developmental stages: egg, blastula and early (e‐)/late (l‐) gastrula stages in lateral views of sagittal sections; e‐neurula and e‐tailbud stages show anterior views (left picture) or dorsal views (right picture, anterior to the left). Mid (m‐) and l‐tailbud stages are shown in lateral views (anterior left). Abbreviations: ba, branchial arches; cnc, cranial neural crest; eye, retinal Anlage; nf, neural folds; ov, otic vesicle. Top: Targeting region of the pwMO oligonucleotide on the X. laevis pwwp2a mRNA. The translational start site is highlighted in red. To determine the translational blocking efficiency of pwMO in vivo , a ˜275‐bp fragment of pwwp2a 5′ cDNA sequence, including the AUG, was cloned in frame upstream of the luciferase coding region. Bottom: The above depicted luciferase construct was injected in CoMO‐ or pwMO‐loaded embryos and chemiluminescence measured at gastrula stage. Error bars indicate SEM of three independent biological replicates. UI, uninjected embryos. Morphology of X. tropicalis embryos injected with CoMO and pwMO together with lineage‐tracer Alexa‐488; numbers indicate penetrance of major morphological phenotype over total embryos inspected ( n = 3 experiments).
    Figure Legend Snippet: Analysis of Xenopus pwwp2a expression and function Expression pattern of endogenous Xenopus tropicalis pwwp2a mRNA during early development. Pictures show whole‐mount RNA in situ hybridization patterns (purple color) for the following developmental stages: egg, blastula and early (e‐)/late (l‐) gastrula stages in lateral views of sagittal sections; e‐neurula and e‐tailbud stages show anterior views (left picture) or dorsal views (right picture, anterior to the left). Mid (m‐) and l‐tailbud stages are shown in lateral views (anterior left). Abbreviations: ba, branchial arches; cnc, cranial neural crest; eye, retinal Anlage; nf, neural folds; ov, otic vesicle. Top: Targeting region of the pwMO oligonucleotide on the X. laevis pwwp2a mRNA. The translational start site is highlighted in red. To determine the translational blocking efficiency of pwMO in vivo , a ˜275‐bp fragment of pwwp2a 5′ cDNA sequence, including the AUG, was cloned in frame upstream of the luciferase coding region. Bottom: The above depicted luciferase construct was injected in CoMO‐ or pwMO‐loaded embryos and chemiluminescence measured at gastrula stage. Error bars indicate SEM of three independent biological replicates. UI, uninjected embryos. Morphology of X. tropicalis embryos injected with CoMO and pwMO together with lineage‐tracer Alexa‐488; numbers indicate penetrance of major morphological phenotype over total embryos inspected ( n = 3 experiments).

    Techniques Used: Expressing, RNA In Situ Hybridization, Blocking Assay, In Vivo, Sequencing, Clone Assay, Luciferase, Construct, Injection

    18) Product Images from "The lupus susceptibility locus Sgp3 encodes the suppressor of endogenous retrovirus expression SNERV"

    Article Title: The lupus susceptibility locus Sgp3 encodes the suppressor of endogenous retrovirus expression SNERV

    Journal: Immunity

    doi: 10.1016/j.immuni.2018.12.022

    NEERV transcription is globally increased in C57BL/6N, but not C57BL/6J, lymphocytes and bone marrow-derived macrophages (A) Representative histogram and calculated MFI of ERV envelope protein expression detected via FACS on the surface of peripheral blood B cells, CD4 + T, and CD8 + T lymphocytes from adult C57BL/6N (B6N) and C57BL/6J (B6J) mice. Each histogram or point represents an individual mouse and mean and standard deviation are plotted. (B) RT-qPCR of RNA from total splenocytes from B6N (n=8) and B6J (n=8) mice. Primers amplify respective envelope regions of all Xmv, Pmv, Mpmv, and Emv transcripts, the gag or polymerase regions of IAP, MusD, and ETn elements (Maksakova et al., 2009), or LINE1 ORFp1. Values were normalized to GAPDH expression. Mean and standard deviation are plotted. (C) Volcano plot of differentially expressed cellular genes all 47 uniquely mappable ERV loci from mRNA sequencing of B6N and B6J naïve CD4 + T cells. (D) Normalized read counts mapping to NEERV LTR families using the RepEnrich alignment strategy from mRNA sequencing of naïve CD4+ T cells. (E) Volcano plot of differentially expressed cellular genes all 47 uniquely mappable ERV loci from mRNA sequencing of B6N and B6J bone marrow-derived macrophages (F) .
    Figure Legend Snippet: NEERV transcription is globally increased in C57BL/6N, but not C57BL/6J, lymphocytes and bone marrow-derived macrophages (A) Representative histogram and calculated MFI of ERV envelope protein expression detected via FACS on the surface of peripheral blood B cells, CD4 + T, and CD8 + T lymphocytes from adult C57BL/6N (B6N) and C57BL/6J (B6J) mice. Each histogram or point represents an individual mouse and mean and standard deviation are plotted. (B) RT-qPCR of RNA from total splenocytes from B6N (n=8) and B6J (n=8) mice. Primers amplify respective envelope regions of all Xmv, Pmv, Mpmv, and Emv transcripts, the gag or polymerase regions of IAP, MusD, and ETn elements (Maksakova et al., 2009), or LINE1 ORFp1. Values were normalized to GAPDH expression. Mean and standard deviation are plotted. (C) Volcano plot of differentially expressed cellular genes all 47 uniquely mappable ERV loci from mRNA sequencing of B6N and B6J naïve CD4 + T cells. (D) Normalized read counts mapping to NEERV LTR families using the RepEnrich alignment strategy from mRNA sequencing of naïve CD4+ T cells. (E) Volcano plot of differentially expressed cellular genes all 47 uniquely mappable ERV loci from mRNA sequencing of B6N and B6J bone marrow-derived macrophages (F) .

    Techniques Used: Derivative Assay, Expressing, FACS, Mouse Assay, Standard Deviation, Quantitative RT-PCR, Sequencing

    19) Product Images from "Distinct Transcriptional and Alternative Splicing Signatures of Decidual CD4+ T Cells in Early Human Pregnancy"

    Article Title: Distinct Transcriptional and Alternative Splicing Signatures of Decidual CD4+ T Cells in Early Human Pregnancy

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00682

    Human dCD4 T cells show a distinct transcriptional signature and upregulate genes related to immune system process as compared with autologous pCD4 T cells. Three healthy women at the first trimester of normal pregnancy were recruited and their dCD4 and pCD4 T cells were isolated by fluorescence-activated cell sorting (FACS). (A) Summary of mRNA-Seq data for the purified dCD4 and pCD4 T cells. RNA samples of paired dCD4 and pCD4 T cells from three individuals were sequenced on the Illumina Hiseq 2500 platform, yielding approximately 30–40 million 2 × 125-bp paired-end reads per sample, which were then mapped to the human reference genome (hg19 version). (B) Number and percentage of the differentially expressed genes ( P
    Figure Legend Snippet: Human dCD4 T cells show a distinct transcriptional signature and upregulate genes related to immune system process as compared with autologous pCD4 T cells. Three healthy women at the first trimester of normal pregnancy were recruited and their dCD4 and pCD4 T cells were isolated by fluorescence-activated cell sorting (FACS). (A) Summary of mRNA-Seq data for the purified dCD4 and pCD4 T cells. RNA samples of paired dCD4 and pCD4 T cells from three individuals were sequenced on the Illumina Hiseq 2500 platform, yielding approximately 30–40 million 2 × 125-bp paired-end reads per sample, which were then mapped to the human reference genome (hg19 version). (B) Number and percentage of the differentially expressed genes ( P

    Techniques Used: Isolation, Fluorescence, FACS, Purification

    20) Product Images from "SUMOylation of the m6A-RNA methyltransferase METTL3 modulates its function"

    Article Title: SUMOylation of the m6A-RNA methyltransferase METTL3 modulates its function

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky156

    SUMO1 modification of METTL3 represses its RNA m 6 A methyltransferase activity. (A–E) Polyadenylated mRNAs were purified for the dot-blot assay (upper panels), and cell lysates were used for immunoblotting with indicated antibodies (lower panels). ( A ) METTL3 is a main component responsible for the abundance of m 6 A in mRNAs. The abundance of m 6 A in mRNAs from shControl or shMETTL3 293T and H1299 cells was detected by the Dot-blot assay with anti-m 6 A antibody, and equal loading of the mRNAs was verified by methylene blue staining (upper panels). METTL3 knockdown efficiency in 293T and H1299 cells was shown (lower panels). ( B ) The level of m 6 A in mRNAs is low in the high SUMOylation status in SENP1 knockdown cells. ( C ) SUMOylation of METTL3 reduces its m 6 A methyltransferase activity. HA-METTL3 with or without His-SUMO1/Flag-Ubc9 were transfected into 293T cells. (D–F) The SUMO-site mutataion 4KR (K 177/211/212/215 R) of METTL3 significantly enhances its m 6 A methyltransferase activity. ( D ) HA-METTL3-WT or -4KR was transiently transfeced into 293T cells, and ( E ) HA-METTL3-WT or -4KR was stably re-expressed H1299-shMETTL3 by using the lentiviral system. ( F ) HA-METTL3-WT or -4KR were transfected with or without His-SUMO1/Flag-Ubc9 into 293T cells. The SUMOylation assays and dot-blot assays were performed as described before. ( G ) LC–MS/MS quantification of the m 6 A/A ratio in polyadenylated RNAs purified from H1299-shMETTL3 cells with METTL3-WT or METTL3-4KR. Error bars indicate mean ± S.D. (two technical replicates). ( H ) The in vitro RNA N6-adenosine methylation activity was tested using purified Flag-METTL3-WT, SUMOlated Flag-METTL3-WT or Flag-METTL3-4KR proteins in combination with purified Flag-METTL14 and RNA-probe (Seq1) with consensus sequence of ‘GGACU’. The methylation of RNA-probe was measured by immunoblotting with the m 6 A antibody.
    Figure Legend Snippet: SUMO1 modification of METTL3 represses its RNA m 6 A methyltransferase activity. (A–E) Polyadenylated mRNAs were purified for the dot-blot assay (upper panels), and cell lysates were used for immunoblotting with indicated antibodies (lower panels). ( A ) METTL3 is a main component responsible for the abundance of m 6 A in mRNAs. The abundance of m 6 A in mRNAs from shControl or shMETTL3 293T and H1299 cells was detected by the Dot-blot assay with anti-m 6 A antibody, and equal loading of the mRNAs was verified by methylene blue staining (upper panels). METTL3 knockdown efficiency in 293T and H1299 cells was shown (lower panels). ( B ) The level of m 6 A in mRNAs is low in the high SUMOylation status in SENP1 knockdown cells. ( C ) SUMOylation of METTL3 reduces its m 6 A methyltransferase activity. HA-METTL3 with or without His-SUMO1/Flag-Ubc9 were transfected into 293T cells. (D–F) The SUMO-site mutataion 4KR (K 177/211/212/215 R) of METTL3 significantly enhances its m 6 A methyltransferase activity. ( D ) HA-METTL3-WT or -4KR was transiently transfeced into 293T cells, and ( E ) HA-METTL3-WT or -4KR was stably re-expressed H1299-shMETTL3 by using the lentiviral system. ( F ) HA-METTL3-WT or -4KR were transfected with or without His-SUMO1/Flag-Ubc9 into 293T cells. The SUMOylation assays and dot-blot assays were performed as described before. ( G ) LC–MS/MS quantification of the m 6 A/A ratio in polyadenylated RNAs purified from H1299-shMETTL3 cells with METTL3-WT or METTL3-4KR. Error bars indicate mean ± S.D. (two technical replicates). ( H ) The in vitro RNA N6-adenosine methylation activity was tested using purified Flag-METTL3-WT, SUMOlated Flag-METTL3-WT or Flag-METTL3-4KR proteins in combination with purified Flag-METTL14 and RNA-probe (Seq1) with consensus sequence of ‘GGACU’. The methylation of RNA-probe was measured by immunoblotting with the m 6 A antibody.

    Techniques Used: Modification, Activity Assay, Purification, Dot Blot, Staining, Transfection, Stable Transfection, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, In Vitro, Methylation, Sequencing

    SUMOylation of METTL3 down-regulates m 6 A modification in mRNAs resulting in the alternation of gene expression profile. ( A ) Cumulative distribution curve for the abundance of m 6 A modification across the transcriptome of H1299-shMETTL3 cells re-expressing METTL3-WT or METTL3-4KR. ( B ) Distribution of m 6 A peaks across around stop codons and 3′ UTRs of the entire set of mRNA transcripts. ( C ) Comparison of the abundance of m 6 A peaks across the transcriptome of H1299-shMETTL3 cells re-expressing METTL3-WT or METTL3-4KR. The fold-change ≥2.0 was considered to be significant, which was the abundance of m 6 A peaks of METTL3-4KR relative to METTL3-WT. IP/Input, was referred to as the abundance of m 6 A peak in mRNAs detected in MeRIP m 6 A-Seq (IP) normalized by that detected in RNA-Seq (Input). ( D ) Heatmap showing the alternation of mRNA expression profiles in H1299-shMETTL3 cells re-expressing METTL3-WT or METTL3-4KR.
    Figure Legend Snippet: SUMOylation of METTL3 down-regulates m 6 A modification in mRNAs resulting in the alternation of gene expression profile. ( A ) Cumulative distribution curve for the abundance of m 6 A modification across the transcriptome of H1299-shMETTL3 cells re-expressing METTL3-WT or METTL3-4KR. ( B ) Distribution of m 6 A peaks across around stop codons and 3′ UTRs of the entire set of mRNA transcripts. ( C ) Comparison of the abundance of m 6 A peaks across the transcriptome of H1299-shMETTL3 cells re-expressing METTL3-WT or METTL3-4KR. The fold-change ≥2.0 was considered to be significant, which was the abundance of m 6 A peaks of METTL3-4KR relative to METTL3-WT. IP/Input, was referred to as the abundance of m 6 A peak in mRNAs detected in MeRIP m 6 A-Seq (IP) normalized by that detected in RNA-Seq (Input). ( D ) Heatmap showing the alternation of mRNA expression profiles in H1299-shMETTL3 cells re-expressing METTL3-WT or METTL3-4KR.

    Techniques Used: Modification, Expressing, RNA Sequencing Assay

    21) Product Images from "Finding Nemo: hybrid assembly with Oxford Nanopore and Illumina reads greatly improves the clownfish (Amphiprion ocellaris) genome assembly"

    Article Title: Finding Nemo: hybrid assembly with Oxford Nanopore and Illumina reads greatly improves the clownfish (Amphiprion ocellaris) genome assembly

    Journal: GigaScience

    doi: 10.1093/gigascience/gix137

    Mapping of MinION long reads, Illumina-assembled scaffolds, and RNA-sequencing reads of male and female A. ocellaris to the genomic region containing the cyp19a1a ].
    Figure Legend Snippet: Mapping of MinION long reads, Illumina-assembled scaffolds, and RNA-sequencing reads of male and female A. ocellaris to the genomic region containing the cyp19a1a ].

    Techniques Used: RNA Sequencing Assay

    22) Product Images from "Enhanced Anti-lymphoma Activity of CAR19-iNKT Cells Underpinned by Dual CD19 and CD1d Targeting"

    Article Title: Enhanced Anti-lymphoma Activity of CAR19-iNKT Cells Underpinned by Dual CD19 and CD1d Targeting

    Journal: Cancer Cell

    doi: 10.1016/j.ccell.2018.08.017

    Transcriptional and Epigenetic Basis for Enhancing CAR19-iNKT Cell Reactivity (A) CD1D mRNA quantification by qPCR in CLL cells from two patients upon ATRA treatment (10 −6 M) for 0–96 hr. (B and C) Bar charts (B) and flow cytometry histograms (C) showing CD1d expression on malignant B cells upon ATRA treatment and mean fluorescent intensity (MFI) analysis of CD1d expression in comparison with isotype control. (D) Cytotoxicity of second- and third-generation CAR19-T and -NKT cells against αGalCer-pulsed CLL cells pre-treated with 0.1% DMSO control or 10 −6 M ATRA. Error bars represent SEM of triplicate assays. (E) ChIP-qPCR assay for H3K4me3 and H3K27me3 enrichment in the promoter of CD1D using IgG as control in U266 cells. GAPDH is an active gene control, while HOXA2 is a repressed gene control. ChIP data are shown as a percentage of the input chromatin. (F) ChIP-re-ChIP qPCR assay showing fold enrichment of H3K27me3 or IgG control after immunoprecipitation (IP) against H3K4me3. (G) ChIP-qPCR assay against RNA Pol II for Ser5 over Ser2 phosphorylated form at the promoter of the indicated genes. (H) ChIP-qPCR assay against RARα, EZH2, and Ig control at the promoters of the genes shown. (I) ChIP-re-ChIP qPCR assay showing enrichment of EZH2 or IgG control after IP against RARα in U266 cells for –(I) (n = 3). (J) qPCR quantification of CD1D mRNA in U266 cells treated with 0.1% DMSO, 10 −6 M GSK343, 10 −6 M ATRA or 10 −6 M GSK343 plus 10 −6 M ATRA. Values are normalized to CD1D mRNA expression levels in normal peripheral PB B cells (n = 3). ND, not detectable. (K and L) Relative MFI analysis (K) and histogram depiction (L) of CD1d expression in comparison with isotype control in U266 cells from the same experiment shown in (J). .
    Figure Legend Snippet: Transcriptional and Epigenetic Basis for Enhancing CAR19-iNKT Cell Reactivity (A) CD1D mRNA quantification by qPCR in CLL cells from two patients upon ATRA treatment (10 −6 M) for 0–96 hr. (B and C) Bar charts (B) and flow cytometry histograms (C) showing CD1d expression on malignant B cells upon ATRA treatment and mean fluorescent intensity (MFI) analysis of CD1d expression in comparison with isotype control. (D) Cytotoxicity of second- and third-generation CAR19-T and -NKT cells against αGalCer-pulsed CLL cells pre-treated with 0.1% DMSO control or 10 −6 M ATRA. Error bars represent SEM of triplicate assays. (E) ChIP-qPCR assay for H3K4me3 and H3K27me3 enrichment in the promoter of CD1D using IgG as control in U266 cells. GAPDH is an active gene control, while HOXA2 is a repressed gene control. ChIP data are shown as a percentage of the input chromatin. (F) ChIP-re-ChIP qPCR assay showing fold enrichment of H3K27me3 or IgG control after immunoprecipitation (IP) against H3K4me3. (G) ChIP-qPCR assay against RNA Pol II for Ser5 over Ser2 phosphorylated form at the promoter of the indicated genes. (H) ChIP-qPCR assay against RARα, EZH2, and Ig control at the promoters of the genes shown. (I) ChIP-re-ChIP qPCR assay showing enrichment of EZH2 or IgG control after IP against RARα in U266 cells for –(I) (n = 3). (J) qPCR quantification of CD1D mRNA in U266 cells treated with 0.1% DMSO, 10 −6 M GSK343, 10 −6 M ATRA or 10 −6 M GSK343 plus 10 −6 M ATRA. Values are normalized to CD1D mRNA expression levels in normal peripheral PB B cells (n = 3). ND, not detectable. (K and L) Relative MFI analysis (K) and histogram depiction (L) of CD1d expression in comparison with isotype control in U266 cells from the same experiment shown in (J). .

    Techniques Used: Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Expressing, Chromatin Immunoprecipitation, Immunoprecipitation

    23) Product Images from "The transcription factor Foxo1 controls germinal center B cell proliferation in response to T cell help"

    Article Title: The transcription factor Foxo1 controls germinal center B cell proliferation in response to T cell help

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20161263

    Foxo1 is required for GC maintenance. (A) Schematic illustration of the experimental protocol for B–D, F, and G. (B) Flow cytometry of NP-specific donor B cells (CD45.1 + B220 + NP + ). (C) Histograms representing the number of donor IgG1 − GC B cells (CD45.1 + B220 + NP + CD38 − IgG1 − ) and IgG1 + GC B cells (CD45.1 + B220 + NP + CD38 − IgG1 + ) in 10 6 splenocytes (left), and the ratio of DZ:LZ cells (right). n = 3 biological replicates. (D, left) DNA content measurement of Foxo1 +/+ and Foxo1 f/f LZ GC B cells assessed by 7-AAD staining. n = 5 and 3 biological replicates for tamoxifen and vehicle treatment, respectively. (right) Proliferation status of Foxo1 +/+ and Foxo1 f/f LZ GC B cells assessed by EdU incorporation 30 min after an EdU injection. n = 3 biological replicates. (E) Immunohistochemical analysis. (top) Schematic illustration of the experimental protocol. (bottom left) Representative images of immunofluorescence microscopy of spleen sections showing expression of CD45.1 ( Foxo1 f/f -derived donor cells), CD35 (FDC network), and IgD (follicular B cells). DZ and LZ defined by the presence of CD35 + FDCs are surrounded by dashed lines. Bars, 100 µm. (bottom right) Quantification of relative CD45.1 signal intensity in the DZ compared with that in the LZ. Each symbol represents a single GC, and red bars indicate the mean. n = 43 (tamoxifen) and 40 (vehicle) GC pooled from three animals. (F) Hierarchical clustering of the gene expression profiles of Foxo1 +/+ DZ, Foxo1 f/f GC, and Foxo1 +/+ LZ B cells using genes differentially expressed (more than twofold) between Foxo1 +/+ DZ and Foxo1 +/+ LZ B cells (normalized log 2 values based on RNA-seq analysis). n = 3 biological replicates. (G) Gene set enrichment analysis showing the enrichment for genes up-regulated after ligation of CD40 (top) and BCR (bottom) compared of Foxo1 +/+ LZ B cells with Foxo1 f/f GC B cells. Error bars represent SD. Data are representative of three (B and C) or two independent experiments (D and E) and from one experiment (F and G). **, P
    Figure Legend Snippet: Foxo1 is required for GC maintenance. (A) Schematic illustration of the experimental protocol for B–D, F, and G. (B) Flow cytometry of NP-specific donor B cells (CD45.1 + B220 + NP + ). (C) Histograms representing the number of donor IgG1 − GC B cells (CD45.1 + B220 + NP + CD38 − IgG1 − ) and IgG1 + GC B cells (CD45.1 + B220 + NP + CD38 − IgG1 + ) in 10 6 splenocytes (left), and the ratio of DZ:LZ cells (right). n = 3 biological replicates. (D, left) DNA content measurement of Foxo1 +/+ and Foxo1 f/f LZ GC B cells assessed by 7-AAD staining. n = 5 and 3 biological replicates for tamoxifen and vehicle treatment, respectively. (right) Proliferation status of Foxo1 +/+ and Foxo1 f/f LZ GC B cells assessed by EdU incorporation 30 min after an EdU injection. n = 3 biological replicates. (E) Immunohistochemical analysis. (top) Schematic illustration of the experimental protocol. (bottom left) Representative images of immunofluorescence microscopy of spleen sections showing expression of CD45.1 ( Foxo1 f/f -derived donor cells), CD35 (FDC network), and IgD (follicular B cells). DZ and LZ defined by the presence of CD35 + FDCs are surrounded by dashed lines. Bars, 100 µm. (bottom right) Quantification of relative CD45.1 signal intensity in the DZ compared with that in the LZ. Each symbol represents a single GC, and red bars indicate the mean. n = 43 (tamoxifen) and 40 (vehicle) GC pooled from three animals. (F) Hierarchical clustering of the gene expression profiles of Foxo1 +/+ DZ, Foxo1 f/f GC, and Foxo1 +/+ LZ B cells using genes differentially expressed (more than twofold) between Foxo1 +/+ DZ and Foxo1 +/+ LZ B cells (normalized log 2 values based on RNA-seq analysis). n = 3 biological replicates. (G) Gene set enrichment analysis showing the enrichment for genes up-regulated after ligation of CD40 (top) and BCR (bottom) compared of Foxo1 +/+ LZ B cells with Foxo1 f/f GC B cells. Error bars represent SD. Data are representative of three (B and C) or two independent experiments (D and E) and from one experiment (F and G). **, P

    Techniques Used: Flow Cytometry, Cytometry, Staining, Injection, Immunohistochemistry, Immunofluorescence, Microscopy, Expressing, Derivative Assay, RNA Sequencing Assay, Ligation

    24) Product Images from "SUMOylation of the m6A-RNA methyltransferase METTL3 modulates its function"

    Article Title: SUMOylation of the m6A-RNA methyltransferase METTL3 modulates its function

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky156

    SUMO1 modification of METTL3 represses its RNA m 6 A methyltransferase activity. (A–E) Polyadenylated mRNAs were purified for the dot-blot assay (upper panels), and cell lysates were used for immunoblotting with indicated antibodies (lower panels). ( A ) METTL3 is a main component responsible for the abundance of m 6 A in mRNAs. The abundance of m 6 A in mRNAs from shControl or shMETTL3 293T and H1299 cells was detected by the Dot-blot assay with anti-m 6 A antibody, and equal loading of the mRNAs was verified by methylene blue staining (upper panels). METTL3 knockdown efficiency in 293T and H1299 cells was shown (lower panels). ( B ) The level of m 6 A in mRNAs is low in the high SUMOylation status in SENP1 knockdown cells. ( C ) SUMOylation of METTL3 reduces its m 6 A methyltransferase activity. HA-METTL3 with or without His-SUMO1/Flag-Ubc9 were transfected into 293T cells. (D–F) The SUMO-site mutataion 4KR (K 177/211/212/215 R) of METTL3 significantly enhances its m 6 A methyltransferase activity. ( D ) HA-METTL3-WT or -4KR was transiently transfeced into 293T cells, and ( E ) HA-METTL3-WT or -4KR was stably re-expressed H1299-shMETTL3 by using the lentiviral system. ( F ) HA-METTL3-WT or -4KR were transfected with or without His-SUMO1/Flag-Ubc9 into 293T cells. The SUMOylation assays and dot-blot assays were performed as described before. ( G ) LC–MS/MS quantification of the m 6 A/A ratio in polyadenylated RNAs purified from H1299-shMETTL3 cells with METTL3-WT or METTL3-4KR. Error bars indicate mean ± S.D. (two technical replicates). ( H ) The in vitro RNA N6-adenosine methylation activity was tested using purified Flag-METTL3-WT, SUMOlated Flag-METTL3-WT or Flag-METTL3-4KR proteins in combination with purified Flag-METTL14 and RNA-probe (Seq1) with consensus sequence of ‘GGACU’. The methylation of RNA-probe was measured by immunoblotting with the m 6 A antibody.
    Figure Legend Snippet: SUMO1 modification of METTL3 represses its RNA m 6 A methyltransferase activity. (A–E) Polyadenylated mRNAs were purified for the dot-blot assay (upper panels), and cell lysates were used for immunoblotting with indicated antibodies (lower panels). ( A ) METTL3 is a main component responsible for the abundance of m 6 A in mRNAs. The abundance of m 6 A in mRNAs from shControl or shMETTL3 293T and H1299 cells was detected by the Dot-blot assay with anti-m 6 A antibody, and equal loading of the mRNAs was verified by methylene blue staining (upper panels). METTL3 knockdown efficiency in 293T and H1299 cells was shown (lower panels). ( B ) The level of m 6 A in mRNAs is low in the high SUMOylation status in SENP1 knockdown cells. ( C ) SUMOylation of METTL3 reduces its m 6 A methyltransferase activity. HA-METTL3 with or without His-SUMO1/Flag-Ubc9 were transfected into 293T cells. (D–F) The SUMO-site mutataion 4KR (K 177/211/212/215 R) of METTL3 significantly enhances its m 6 A methyltransferase activity. ( D ) HA-METTL3-WT or -4KR was transiently transfeced into 293T cells, and ( E ) HA-METTL3-WT or -4KR was stably re-expressed H1299-shMETTL3 by using the lentiviral system. ( F ) HA-METTL3-WT or -4KR were transfected with or without His-SUMO1/Flag-Ubc9 into 293T cells. The SUMOylation assays and dot-blot assays were performed as described before. ( G ) LC–MS/MS quantification of the m 6 A/A ratio in polyadenylated RNAs purified from H1299-shMETTL3 cells with METTL3-WT or METTL3-4KR. Error bars indicate mean ± S.D. (two technical replicates). ( H ) The in vitro RNA N6-adenosine methylation activity was tested using purified Flag-METTL3-WT, SUMOlated Flag-METTL3-WT or Flag-METTL3-4KR proteins in combination with purified Flag-METTL14 and RNA-probe (Seq1) with consensus sequence of ‘GGACU’. The methylation of RNA-probe was measured by immunoblotting with the m 6 A antibody.

    Techniques Used: Modification, Activity Assay, Purification, Dot Blot, Staining, Transfection, Stable Transfection, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, In Vitro, Methylation, Sequencing

    SUMOylation of METTL3 down-regulates m 6 A modification in mRNAs resulting in the alternation of gene expression profile. ( A ) Cumulative distribution curve for the abundance of m 6 A modification across the transcriptome of H1299-shMETTL3 cells re-expressing METTL3-WT or METTL3-4KR. ( B ) Distribution of m 6 A peaks across around stop codons and 3′ UTRs of the entire set of mRNA transcripts. ( C ) Comparison of the abundance of m 6 A peaks across the transcriptome of H1299-shMETTL3 cells re-expressing METTL3-WT or METTL3-4KR. The fold-change ≥2.0 was considered to be significant, which was the abundance of m 6 A peaks of METTL3-4KR relative to METTL3-WT. IP/Input, was referred to as the abundance of m 6 A peak in mRNAs detected in MeRIP m 6 A-Seq (IP) normalized by that detected in RNA-Seq (Input). ( D ) Heatmap showing the alternation of mRNA expression profiles in H1299-shMETTL3 cells re-expressing METTL3-WT or METTL3-4KR.
    Figure Legend Snippet: SUMOylation of METTL3 down-regulates m 6 A modification in mRNAs resulting in the alternation of gene expression profile. ( A ) Cumulative distribution curve for the abundance of m 6 A modification across the transcriptome of H1299-shMETTL3 cells re-expressing METTL3-WT or METTL3-4KR. ( B ) Distribution of m 6 A peaks across around stop codons and 3′ UTRs of the entire set of mRNA transcripts. ( C ) Comparison of the abundance of m 6 A peaks across the transcriptome of H1299-shMETTL3 cells re-expressing METTL3-WT or METTL3-4KR. The fold-change ≥2.0 was considered to be significant, which was the abundance of m 6 A peaks of METTL3-4KR relative to METTL3-WT. IP/Input, was referred to as the abundance of m 6 A peak in mRNAs detected in MeRIP m 6 A-Seq (IP) normalized by that detected in RNA-Seq (Input). ( D ) Heatmap showing the alternation of mRNA expression profiles in H1299-shMETTL3 cells re-expressing METTL3-WT or METTL3-4KR.

    Techniques Used: Modification, Expressing, RNA Sequencing Assay

    25) Product Images from "DEAD-Box RNA Helicase 42 Plays a Critical Role in Pre-mRNA Splicing under Cold Stress"

    Article Title: DEAD-Box RNA Helicase 42 Plays a Critical Role in Pre-mRNA Splicing under Cold Stress

    Journal: Plant Physiology

    doi: 10.1104/pp.19.00832

    RNA immunoprecipitation analysis for OsRH42. A, RT-PCR and immunoblot analyses for OsRH42-GFP transgenic calli. Total RNA and total soluble proteins were isolated from two independent OsRH42-GFP transgenic lines (42-GFP-1 and 42-GFP-2) and a GFP transgenic line (GFP) and then analyzed via RT-PCR with specific primers for GFP and ACT1 genes and via immunoblotting (WB) with GFP antibody, respectively. B, OsRH42-associated snRNA analysis. Total RNAs and immunoprecipitated (IP) RNAs were subjected to RT-PCR analysis with specific primers for U1, U2, U4, and U6 snRNAs.
    Figure Legend Snippet: RNA immunoprecipitation analysis for OsRH42. A, RT-PCR and immunoblot analyses for OsRH42-GFP transgenic calli. Total RNA and total soluble proteins were isolated from two independent OsRH42-GFP transgenic lines (42-GFP-1 and 42-GFP-2) and a GFP transgenic line (GFP) and then analyzed via RT-PCR with specific primers for GFP and ACT1 genes and via immunoblotting (WB) with GFP antibody, respectively. B, OsRH42-associated snRNA analysis. Total RNAs and immunoprecipitated (IP) RNAs were subjected to RT-PCR analysis with specific primers for U1, U2, U4, and U6 snRNAs.

    Techniques Used: Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Transgenic Assay, Isolation, Western Blot

    26) Product Images from "Evolution of a Designed Protein Assembly Encapsulating its Own RNA Genome"

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

    Journal: Nature

    doi: 10.1038/nature25157

    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
    Figure Legend 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

    Techniques Used: 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.
    Figure Legend 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.

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

    27) Product Images from "Integrated microRNA and mRNA analysis in the pathogenic filamentous fungus Trichophyton rubrum"

    Article Title: Integrated microRNA and mRNA analysis in the pathogenic filamentous fungus Trichophyton rubrum

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-5316-3

    Validation of RNA-Seq results by qRT-PCR. Three biological replicates were performed. * indicates significant difference of milRNA/mRNA expression level in conidial vs. mycelial stages (*: P
    Figure Legend Snippet: Validation of RNA-Seq results by qRT-PCR. Three biological replicates were performed. * indicates significant difference of milRNA/mRNA expression level in conidial vs. mycelial stages (*: P

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

    28) Product Images from "Methods to Determine the Transcriptomes of Trypanosomes in Mixtures with Mammalian Cells: The Effects of Parasite Purification and Selective cDNA Amplification"

    Article Title: Methods to Determine the Transcriptomes of Trypanosomes in Mixtures with Mammalian Cells: The Effects of Parasite Purification and Selective cDNA Amplification

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0002806

    Amplification procedure. For explanation see text. A. RNA extraction; B. Mixing of trypanosome and HeLa RNA, if applicable; C. Location of cDNA primers; D. Location of SL reverse primer; E. Primers used for PCR; F. PCR product; G. Sheared PCR products; H. Sheared DNAs with Illumina adaptors; I. Sequence output; J. Sequences aligned to genome.
    Figure Legend Snippet: Amplification procedure. For explanation see text. A. RNA extraction; B. Mixing of trypanosome and HeLa RNA, if applicable; C. Location of cDNA primers; D. Location of SL reverse primer; E. Primers used for PCR; F. PCR product; G. Sheared PCR products; H. Sheared DNAs with Illumina adaptors; I. Sequence output; J. Sequences aligned to genome.

    Techniques Used: Amplification, RNA Extraction, Polymerase Chain Reaction, Sequencing

    29) Product Images from "Polycomb- and Methylation-Independent Roles of EZH2 as a Transcription Activator"

    Article Title: Polycomb- and Methylation-Independent Roles of EZH2 as a Transcription Activator

    Journal: Cell reports

    doi: 10.1016/j.celrep.2018.11.035

    EZH2 Directly Activates AR Gene Transcription (A) EZH2 protein occupies the AR gene promoter. EZH2 ChIP-seq was performed in LNCaP cells with an antibody targeting endogenous EZH2 (top). HA ChIP-seq was performed using an anti-HA antibody in LNCaP cells with ectopic HA-EZH2 overexpression. Two biological replicates are shown (center and bottom). (B) ChIP-qPCR showing EZH2 binding along the AR gene promoter. ChIP was performed in LNCaP cells using anti-EZH2 and IgG antibodies and then subjected to qPCR using primer pairs targeting ~60-bp sliding windows within −1 kb to +3 kb of the AR gene. The x axis indicates the central location of the PCR products relative to the AR TSS. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (C) Different regions (of 400 bp) of the AR promoter (from 0 to +3 kb) were cloned into the pRetroX-Tight-Pur-Luc vector and transfected into 293T cells, which were then subjected to ChIP by anti-EZH2 or IgG. EZH2 occupancy at the ectopically expressed AR promoter was determined by qPCR using a common forward primer targeting the vector sequence and a reverse primer specific to each fragment. Data shown are mean (±SEM) of technical replicates from one representative experiment of two. (D) Various AR promoter regions were cloned into the pGL4.10 vector and transfected into 293T cells with either control pLVX or HA-EZH2 overexpression. Cells were then subjected to luciferase reporter assays. Results were normalized to the Renilla internal control. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (E) Schematic view of the AR promoter sequence starting from the transcription start site (TSS). The sgRNAs were labeled sgAR1 to 4, their sequences are shown in green font, and their distances to the AR TSS are marked as numbers. The primers (F2 and R2) for PCR validation are shown in purple. (F and G) The distal AR promoter region is required for EZH2 activation of AR transcription. LNCaP cells were infected with lentiCRISPR-Cas9 containing the pLENTI.V2 control, sgAR1+2, sgAR3+4, or sgAR1+4 for 48 hr. CRISPR-Cas9-mediated genome editing was confirmed by Sanger sequencing (F) and genomic DNA PCR (G) using primers F2 and R2 (indicated in A and E). (H) CRISPR-Cas9-edited LNCaP cells were transfected with control or EZH2-targeting siRNA for 48 hr. Total RNA was harvested and subjected to RT-PCR analysis using F2 and R2, which are expected to yield a wild-type (AR WT, top band with black asterisk) and a CRISPR-Cas9-deleted (AR del, bottom bands with red asterisk) AR mRNA.
    Figure Legend Snippet: EZH2 Directly Activates AR Gene Transcription (A) EZH2 protein occupies the AR gene promoter. EZH2 ChIP-seq was performed in LNCaP cells with an antibody targeting endogenous EZH2 (top). HA ChIP-seq was performed using an anti-HA antibody in LNCaP cells with ectopic HA-EZH2 overexpression. Two biological replicates are shown (center and bottom). (B) ChIP-qPCR showing EZH2 binding along the AR gene promoter. ChIP was performed in LNCaP cells using anti-EZH2 and IgG antibodies and then subjected to qPCR using primer pairs targeting ~60-bp sliding windows within −1 kb to +3 kb of the AR gene. The x axis indicates the central location of the PCR products relative to the AR TSS. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (C) Different regions (of 400 bp) of the AR promoter (from 0 to +3 kb) were cloned into the pRetroX-Tight-Pur-Luc vector and transfected into 293T cells, which were then subjected to ChIP by anti-EZH2 or IgG. EZH2 occupancy at the ectopically expressed AR promoter was determined by qPCR using a common forward primer targeting the vector sequence and a reverse primer specific to each fragment. Data shown are mean (±SEM) of technical replicates from one representative experiment of two. (D) Various AR promoter regions were cloned into the pGL4.10 vector and transfected into 293T cells with either control pLVX or HA-EZH2 overexpression. Cells were then subjected to luciferase reporter assays. Results were normalized to the Renilla internal control. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (E) Schematic view of the AR promoter sequence starting from the transcription start site (TSS). The sgRNAs were labeled sgAR1 to 4, their sequences are shown in green font, and their distances to the AR TSS are marked as numbers. The primers (F2 and R2) for PCR validation are shown in purple. (F and G) The distal AR promoter region is required for EZH2 activation of AR transcription. LNCaP cells were infected with lentiCRISPR-Cas9 containing the pLENTI.V2 control, sgAR1+2, sgAR3+4, or sgAR1+4 for 48 hr. CRISPR-Cas9-mediated genome editing was confirmed by Sanger sequencing (F) and genomic DNA PCR (G) using primers F2 and R2 (indicated in A and E). (H) CRISPR-Cas9-edited LNCaP cells were transfected with control or EZH2-targeting siRNA for 48 hr. Total RNA was harvested and subjected to RT-PCR analysis using F2 and R2, which are expected to yield a wild-type (AR WT, top band with black asterisk) and a CRISPR-Cas9-deleted (AR del, bottom bands with red asterisk) AR mRNA.

    Techniques Used: Chromatin Immunoprecipitation, Over Expression, Real-time Polymerase Chain Reaction, Binding Assay, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Transfection, Sequencing, Luciferase, Labeling, Activation Assay, Infection, CRISPR, Reverse Transcription Polymerase Chain Reaction

    30) Product Images from "An Inversion Disrupting FAM134B Is Associated with Sensory Neuropathy in the Border Collie Dog Breed"

    Article Title: An Inversion Disrupting FAM134B Is Associated with Sensory Neuropathy in the Border Collie Dog Breed

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.116.027896

    Example of novel exon formation through cryptic splicing. An example of a novel exon occurring before an inversion breakpoint due to a cryptic splicing event. The novel splice acceptor site is located upstream of inversion breakpoint. Two further novel splice acceptor sites are located within the inverted region. Transcription of exons 4 to 9 of FAM134B is abolished in the SN case due to relocation of exons 1 to 3 through the inversion event. Note: The control RNAseq dataset is from cerebellum and is shown to illustrate a normal FAM134B splicing pattern. The choice of control tissue was based on availability. Chr4, chromosome 4; RNAseq, RNA sequencing; SN, sensory neuropathy
    Figure Legend Snippet: Example of novel exon formation through cryptic splicing. An example of a novel exon occurring before an inversion breakpoint due to a cryptic splicing event. The novel splice acceptor site is located upstream of inversion breakpoint. Two further novel splice acceptor sites are located within the inverted region. Transcription of exons 4 to 9 of FAM134B is abolished in the SN case due to relocation of exons 1 to 3 through the inversion event. Note: The control RNAseq dataset is from cerebellum and is shown to illustrate a normal FAM134B splicing pattern. The choice of control tissue was based on availability. Chr4, chromosome 4; RNAseq, RNA sequencing; SN, sensory neuropathy

    Techniques Used: RNA Sequencing Assay

    31) Product Images from "Integrated microRNA and mRNA analysis in the pathogenic filamentous fungus Trichophyton rubrum"

    Article Title: Integrated microRNA and mRNA analysis in the pathogenic filamentous fungus Trichophyton rubrum

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-5316-3

    Validation of RNA-Seq results by qRT-PCR. Three biological replicates were performed. * indicates significant difference of milRNA/mRNA expression level in conidial vs. mycelial stages (*: P
    Figure Legend Snippet: Validation of RNA-Seq results by qRT-PCR. Three biological replicates were performed. * indicates significant difference of milRNA/mRNA expression level in conidial vs. mycelial stages (*: P

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

    32) Product Images from "Human NK cell development in hIL-7 and hIL-15 knockin NOD/SCID/IL2rgKO mice"

    Article Title: Human NK cell development in hIL-7 and hIL-15 knockin NOD/SCID/IL2rgKO mice

    Journal: Life Science Alliance

    doi: 10.26508/lsa.201800195

    Gene expression signature of NK cells in NSG hL-7xhIL-15 humanized mice. RNA sequencing was performed using RNA extracted from hCD56+ splenic NK cells of conventional NSG (NSG, n = 2) and hIL-7xhIL-15 KI NSG (hIL7xhIL-15 KI NSG, n = 6) humanized mice. (A) Differentially expressing genes are shown. Gene expression profiles of human NK cells of humanized mice were compared with those from NK cells recovered from human PBMCs. (B) Expression of genes related to cytokine and cytotoxicity are shown.
    Figure Legend Snippet: Gene expression signature of NK cells in NSG hL-7xhIL-15 humanized mice. RNA sequencing was performed using RNA extracted from hCD56+ splenic NK cells of conventional NSG (NSG, n = 2) and hIL-7xhIL-15 KI NSG (hIL7xhIL-15 KI NSG, n = 6) humanized mice. (A) Differentially expressing genes are shown. Gene expression profiles of human NK cells of humanized mice were compared with those from NK cells recovered from human PBMCs. (B) Expression of genes related to cytokine and cytotoxicity are shown.

    Techniques Used: Expressing, Mouse Assay, RNA Sequencing Assay

    33) Product Images from "BMPs as new insulin sensitizers: enhanced glucose uptake in mature 3T3-L1 adipocytes via PPARγ and GLUT4 upregulation"

    Article Title: BMPs as new insulin sensitizers: enhanced glucose uptake in mature 3T3-L1 adipocytes via PPARγ and GLUT4 upregulation

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-17595-5

    BMP2 and BMP6 stimulation regulate mRNA levels of metabolic enzymes/transporters and visfatin. Validation of genes responding to BMP stimulation by qRT-PCR. Adipocytes were treated as outlined in Fig. 2 . Data are presented as means + SEM of two independent experiments different from samples used in the RNAseq experiment, n = 2 for a-c,e-i; n = 3 for d. Lpl ( a ) and Fasn ( b ) were selected from Cluster A. Plin1 ( c ), Id1 ( e ) and Rxra ( f ) and Nampt ( g ) coding for Pre-B-cell colony-enhancing factor 1 (PBEF1) or visfatin were selected from Cluster B. Glut-1 ( d ) was not found to be significantly regulated in the RNA-Seq approach, but selected for validation as it represents the second glucose transporter present in adipocytes. Fatp1 ( h ) and Lep ( i ) were selected from Cluster D. Asteriks denote PPARγ target genes.
    Figure Legend Snippet: BMP2 and BMP6 stimulation regulate mRNA levels of metabolic enzymes/transporters and visfatin. Validation of genes responding to BMP stimulation by qRT-PCR. Adipocytes were treated as outlined in Fig. 2 . Data are presented as means + SEM of two independent experiments different from samples used in the RNAseq experiment, n = 2 for a-c,e-i; n = 3 for d. Lpl ( a ) and Fasn ( b ) were selected from Cluster A. Plin1 ( c ), Id1 ( e ) and Rxra ( f ) and Nampt ( g ) coding for Pre-B-cell colony-enhancing factor 1 (PBEF1) or visfatin were selected from Cluster B. Glut-1 ( d ) was not found to be significantly regulated in the RNA-Seq approach, but selected for validation as it represents the second glucose transporter present in adipocytes. Fatp1 ( h ) and Lep ( i ) were selected from Cluster D. Asteriks denote PPARγ target genes.

    Techniques Used: Quantitative RT-PCR, RNA Sequencing Assay

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    New England Biolabs ultra ii directional rna library prep kit
    Loss of Pnt results in Naa-to-Nab transformations in diverse sensillar subtypes. a A sensillum can contain up to four OSNs through differentiation of Naa (cyan), Nab (magenta), Nba (green), Nbb (yellow) terminal daughter cells originating from a single SOP lineage. b Representative images of <t>RNA</t> FISH for Or67d (magenta) in at1 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or67d-expressing OSNs are duplicated (arrow). A schematic of the proposed Naa-to-Nab fate transformation is shown on the right (color scheme as in ( a )). Scale bar = 2 µm. The open circles in this and other schematics represent OSN precursors that have undergone apoptosis. c Representative images of RNA FISH for Or85a (magenta) and Or59b (green) in ab2 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or85a OSNs (Nab) are duplicated (arrow), while Or59b OSNs (Nba) are unaffected. d Representative images of RNA FISH for Or85b (magenta) and Or22a (green) in ab3 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or85b OSNs (Nab) are duplicated (arrow), while Or22a OSNs (Nba) are unaffected. e Representative images of RNA FISH for Or92a (magenta) and Or10a (cyan) in ab1 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or92a OSNs (Nab) are duplicated (arrow), while Or10a OSNs (Naa) are lost. f Top: theoretical ratios of OSN types in 2-, 3- and 4-neuron sensilla in control and pnt RNAi antennae, assuming Naa-to-Nab fate transformation (i.e. loss of Naa OSNs, and duplication of Nab OSNs). Bottom: experimentally determined OSN ratios in all sensilla in pnt RNAi antennae using as a proxy the normalized ratios of olfactory receptor <t>mRNA</t> expression from antennal transcriptomes (see Supplementary Fig. 6e ). In ab10, Or49a is reported to be coexpressed with Or85f 13 , but transcript levels for this gene were below the cut-off applied during the analysis of these RNA-seq datasets
    Ultra Ii Directional Rna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs nebnext ultra directional rna library prep kit for illumina
    Schematic overview of Hi-GRIL-seq. Induction of T4 <t>RNA</t> ligase expression from the P tac promoter with IPTG leads to the expression of the enzyme and the formation of chimeras between base paired endogenous sRNAs and their targets. Following isolation of total RNA and rRNA depletion, a cDNA library for <t>Illumina</t> sequencing is constructed and sequenced. RNA interactions between sRNAs and their targets are identified by a BLAST-based analysis pipeline. Global chimeras are visualized in a two-dimensional dot plot, in which the location of the dot represents the genomic coordinate of the participating RNAs. To examine the targets of a particular RNA, the coverage of its targets can be visualized. To further zoom in on a particular interaction between the two RNAs, the exact location of ligation junctions in the chimeras are mapped and visualized.
    Nebnext Ultra Directional Rna Library Prep Kit For Illumina, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 474 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Loss of Pnt results in Naa-to-Nab transformations in diverse sensillar subtypes. a A sensillum can contain up to four OSNs through differentiation of Naa (cyan), Nab (magenta), Nba (green), Nbb (yellow) terminal daughter cells originating from a single SOP lineage. b Representative images of RNA FISH for Or67d (magenta) in at1 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or67d-expressing OSNs are duplicated (arrow). A schematic of the proposed Naa-to-Nab fate transformation is shown on the right (color scheme as in ( a )). Scale bar = 2 µm. The open circles in this and other schematics represent OSN precursors that have undergone apoptosis. c Representative images of RNA FISH for Or85a (magenta) and Or59b (green) in ab2 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or85a OSNs (Nab) are duplicated (arrow), while Or59b OSNs (Nba) are unaffected. d Representative images of RNA FISH for Or85b (magenta) and Or22a (green) in ab3 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or85b OSNs (Nab) are duplicated (arrow), while Or22a OSNs (Nba) are unaffected. e Representative images of RNA FISH for Or92a (magenta) and Or10a (cyan) in ab1 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or92a OSNs (Nab) are duplicated (arrow), while Or10a OSNs (Naa) are lost. f Top: theoretical ratios of OSN types in 2-, 3- and 4-neuron sensilla in control and pnt RNAi antennae, assuming Naa-to-Nab fate transformation (i.e. loss of Naa OSNs, and duplication of Nab OSNs). Bottom: experimentally determined OSN ratios in all sensilla in pnt RNAi antennae using as a proxy the normalized ratios of olfactory receptor mRNA expression from antennal transcriptomes (see Supplementary Fig. 6e ). In ab10, Or49a is reported to be coexpressed with Or85f 13 , but transcript levels for this gene were below the cut-off applied during the analysis of these RNA-seq datasets

    Journal: Nature Communications

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

    doi: 10.1038/s41467-019-08345-4

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

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

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

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-019-08345-4

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

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

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

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-019-08345-4

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

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

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

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

    Journal: Molecular microbiology

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

    doi: 10.1111/mmi.13857

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

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

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

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

    Journal: Genes & Development

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

    doi: 10.1101/gad.241950.114

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

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

    Techniques: RNA Sequencing Assay, Derivative Assay