Structured Review

Thermo Fisher rna tags
IRE1 associates with specific surfaces on the ribosome. ( A ) IRE1 crosslink sites do not coincide with published DDX3 crosslink sites on ribosomes (depicted in yellow) ( Oh et al., 2016 ). ( B ) Topographical localization of ribosomal proteins <t>crosslinked</t> to IRE1 through <t>RNA</t> bridges and recovered in denaturing IPs which lie adjacent to the mRNA exit site on the ribosome.
Rna Tags, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rna tags/product/Thermo Fisher
Average 94 stars, based on 3 article reviews
Price from $9.99 to $1999.99
rna tags - by Bioz Stars, 2020-04
94/100 stars

Images

1) Product Images from "The unfolded protein response and endoplasmic reticulum protein targeting machineries converge on the stress sensor IRE1"

Article Title: The unfolded protein response and endoplasmic reticulum protein targeting machineries converge on the stress sensor IRE1

Journal: eLife

doi: 10.7554/eLife.43036

IRE1 associates with specific surfaces on the ribosome. ( A ) IRE1 crosslink sites do not coincide with published DDX3 crosslink sites on ribosomes (depicted in yellow) ( Oh et al., 2016 ). ( B ) Topographical localization of ribosomal proteins crosslinked to IRE1 through RNA bridges and recovered in denaturing IPs which lie adjacent to the mRNA exit site on the ribosome.
Figure Legend Snippet: IRE1 associates with specific surfaces on the ribosome. ( A ) IRE1 crosslink sites do not coincide with published DDX3 crosslink sites on ribosomes (depicted in yellow) ( Oh et al., 2016 ). ( B ) Topographical localization of ribosomal proteins crosslinked to IRE1 through RNA bridges and recovered in denaturing IPs which lie adjacent to the mRNA exit site on the ribosome.

Techniques Used:

Expression profiles of IRE1-associated RNAs identified by PAR-CLIP. ( A ) Left panels: Scatter plots showing the correlation between the number of crosslinked transcript copies (per million reads) recovered by PAR-CLIP and the expression level as measured by RNA-Seq in the HEK293Trex cell line that force-expresses epitope-tagged IRE1, and under the same ER stress conditions (5 μg/ml tunicamycin). The mean expression level for each transcript (obtained from biological duplicates) was normalized to the median expression level of the entire dataset, which consisted of all transcripts for which we recovered RNA-Seq reads (FPKM > 0) in a particular condition. Right panels: Histograms showing the gene expression distribution in RNA-Seq experiments. Deviations from the central tendency are color-coded, with warm colors closer to the central tendency and cool colors towards the tail ends of the distribution. The same color scheme is applied to the data points in the left panels. The dashed vertical lines indicate the standard deviation (SD) of the median-normalized data. ( B ) Waterfall plots showing the expression change in select transcripts identified by PAR-CLIP measured by RNA-Seq in the HEK293Trex cell line that force-expresses epitope-tagged IRE1, and under the same ER stress conditions (5 μg/ml tunicamycin). Bars: mean values of biological duplicates. ( C ) Scatter plots showing the fold-change in the number of crosslinked transcript copies recovered by PAR-CLIP in the presence or absence of chemically induced ER stress and the fold-change in the number of RNA-Seq reads (FPKM) in the same conditions. Genes repressed or expressed during ER stress are indicated on the X-axis. Transcripts for which there is a loss or gain of recovered crosslinked copies are indicated on the Y-axis. ( D ) Representative quantitative real-time PCR measurements of the levels of select PAR-CLIP hit mRNAs in response to ER stress in different cell types. ER stress was induced with 300 nM of the ER calcium reuptake inhibitor thapsigargin, and the cells were pre-treated for 30 min with 5 μg/ml actinomycin D to block transcription. The IRE1 specific inhibitor 4μ8C (50 μM) was used as a control for specificity. Metrics for spliced XBP1 mRNA or the canonical RIDD substrate mRNA, BLOC1S1 , were used as positive controls. Data: mean of triplicates. Error bars: SDs. ( E ) Changes in the transcript levels of select PAR-CLIP targets during ER stress measured by RNA-seq. Data: Mean FPKM values of biological duplicates. Error bars: 95% CI.
Figure Legend Snippet: Expression profiles of IRE1-associated RNAs identified by PAR-CLIP. ( A ) Left panels: Scatter plots showing the correlation between the number of crosslinked transcript copies (per million reads) recovered by PAR-CLIP and the expression level as measured by RNA-Seq in the HEK293Trex cell line that force-expresses epitope-tagged IRE1, and under the same ER stress conditions (5 μg/ml tunicamycin). The mean expression level for each transcript (obtained from biological duplicates) was normalized to the median expression level of the entire dataset, which consisted of all transcripts for which we recovered RNA-Seq reads (FPKM > 0) in a particular condition. Right panels: Histograms showing the gene expression distribution in RNA-Seq experiments. Deviations from the central tendency are color-coded, with warm colors closer to the central tendency and cool colors towards the tail ends of the distribution. The same color scheme is applied to the data points in the left panels. The dashed vertical lines indicate the standard deviation (SD) of the median-normalized data. ( B ) Waterfall plots showing the expression change in select transcripts identified by PAR-CLIP measured by RNA-Seq in the HEK293Trex cell line that force-expresses epitope-tagged IRE1, and under the same ER stress conditions (5 μg/ml tunicamycin). Bars: mean values of biological duplicates. ( C ) Scatter plots showing the fold-change in the number of crosslinked transcript copies recovered by PAR-CLIP in the presence or absence of chemically induced ER stress and the fold-change in the number of RNA-Seq reads (FPKM) in the same conditions. Genes repressed or expressed during ER stress are indicated on the X-axis. Transcripts for which there is a loss or gain of recovered crosslinked copies are indicated on the Y-axis. ( D ) Representative quantitative real-time PCR measurements of the levels of select PAR-CLIP hit mRNAs in response to ER stress in different cell types. ER stress was induced with 300 nM of the ER calcium reuptake inhibitor thapsigargin, and the cells were pre-treated for 30 min with 5 μg/ml actinomycin D to block transcription. The IRE1 specific inhibitor 4μ8C (50 μM) was used as a control for specificity. Metrics for spliced XBP1 mRNA or the canonical RIDD substrate mRNA, BLOC1S1 , were used as positive controls. Data: mean of triplicates. Error bars: SDs. ( E ) Changes in the transcript levels of select PAR-CLIP targets during ER stress measured by RNA-seq. Data: Mean FPKM values of biological duplicates. Error bars: 95% CI.

Techniques Used: Expressing, Cross-linking Immunoprecipitation, RNA Sequencing Assay, Standard Deviation, Real-time Polymerase Chain Reaction, Blocking Assay

A modifed PAR-CLIP protocol for identifying IRE1-associated RNAs. ( A ) Schematic of modified PAR-CLIP protocol. ( B ) Immunoblots showing the enrichment for IRE1 in subcellular fractions prepared by differential detergent extraction as performed in our PAR-CLIP experiments. ( C ) Immunoblot showing the relative enrichment for IRE1 in native and denaturing IP conditions. ( D ) Semi-quantitative RT-PCR showing the extent of XBP1 splicing in the HEK293Trex cell line where we force-express IRE1 in the presence or absence of chemically-induced ER stress. ER stress was induced by exposing the cells to tunicamycin. ( E ) Top: Autoradiogram of an SDS-PAGE gel in which the 5’-ends of IRE1-crosslinked RNAs were radiolabeled with polynucleotide kinase after immunoprecipitation of epitope-tagged IRE1. The crosslinked lysates were treated with the indicated amounts of RNase T1 for 30 min at 25°C. Bottom: Autoradiogram of a TBE-urea PAGE gel in which the RNA fragments purified from gel cutouts obtained from the gel in the top panel were resolved. ( F ) Autoradiogram of an SDS-PAGE gel of the eluates of denaturing IPs of epitope-tagged IRE1 after radiolabeling the 5’-end of crosslinked RNAs. The bracket on the left indicates the region of the gel that was excised for purification of IRE1-RNA complexes. The dashed line indicates where the lanes from a single gel were cropped.
Figure Legend Snippet: A modifed PAR-CLIP protocol for identifying IRE1-associated RNAs. ( A ) Schematic of modified PAR-CLIP protocol. ( B ) Immunoblots showing the enrichment for IRE1 in subcellular fractions prepared by differential detergent extraction as performed in our PAR-CLIP experiments. ( C ) Immunoblot showing the relative enrichment for IRE1 in native and denaturing IP conditions. ( D ) Semi-quantitative RT-PCR showing the extent of XBP1 splicing in the HEK293Trex cell line where we force-express IRE1 in the presence or absence of chemically-induced ER stress. ER stress was induced by exposing the cells to tunicamycin. ( E ) Top: Autoradiogram of an SDS-PAGE gel in which the 5’-ends of IRE1-crosslinked RNAs were radiolabeled with polynucleotide kinase after immunoprecipitation of epitope-tagged IRE1. The crosslinked lysates were treated with the indicated amounts of RNase T1 for 30 min at 25°C. Bottom: Autoradiogram of a TBE-urea PAGE gel in which the RNA fragments purified from gel cutouts obtained from the gel in the top panel were resolved. ( F ) Autoradiogram of an SDS-PAGE gel of the eluates of denaturing IPs of epitope-tagged IRE1 after radiolabeling the 5’-end of crosslinked RNAs. The bracket on the left indicates the region of the gel that was excised for purification of IRE1-RNA complexes. The dashed line indicates where the lanes from a single gel were cropped.

Techniques Used: Cross-linking Immunoprecipitation, Modification, Western Blot, Quantitative RT-PCR, SDS Page, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Purification, Radioactivity

2) Product Images from "The unfolded protein response and endoplasmic reticulum protein targeting machineries converge on the stress sensor IRE1"

Article Title: The unfolded protein response and endoplasmic reticulum protein targeting machineries converge on the stress sensor IRE1

Journal: eLife

doi: 10.7554/eLife.43036

IRE1 associates with specific surfaces on the ribosome. ( A ) IRE1 crosslink sites do not coincide with published DDX3 crosslink sites on ribosomes (depicted in yellow) ( Oh et al., 2016 ). ( B ) Topographical localization of ribosomal proteins crosslinked to IRE1 through RNA bridges and recovered in denaturing IPs which lie adjacent to the mRNA exit site on the ribosome.
Figure Legend Snippet: IRE1 associates with specific surfaces on the ribosome. ( A ) IRE1 crosslink sites do not coincide with published DDX3 crosslink sites on ribosomes (depicted in yellow) ( Oh et al., 2016 ). ( B ) Topographical localization of ribosomal proteins crosslinked to IRE1 through RNA bridges and recovered in denaturing IPs which lie adjacent to the mRNA exit site on the ribosome.

Techniques Used:

Expression profiles of IRE1-associated RNAs identified by PAR-CLIP. ( A ) Left panels: Scatter plots showing the correlation between the number of crosslinked transcript copies (per million reads) recovered by PAR-CLIP and the expression level as measured by RNA-Seq in the HEK293Trex cell line that force-expresses epitope-tagged IRE1, and under the same ER stress conditions (5 μg/ml tunicamycin). The mean expression level for each transcript (obtained from biological duplicates) was normalized to the median expression level of the entire dataset, which consisted of all transcripts for which we recovered RNA-Seq reads (FPKM > 0) in a particular condition. Right panels: Histograms showing the gene expression distribution in RNA-Seq experiments. Deviations from the central tendency are color-coded, with warm colors closer to the central tendency and cool colors towards the tail ends of the distribution. The same color scheme is applied to the data points in the left panels. The dashed vertical lines indicate the standard deviation (SD) of the median-normalized data. ( B ) Waterfall plots showing the expression change in select transcripts identified by PAR-CLIP measured by RNA-Seq in the HEK293Trex cell line that force-expresses epitope-tagged IRE1, and under the same ER stress conditions (5 μg/ml tunicamycin). Bars: mean values of biological duplicates. ( C ) Scatter plots showing the fold-change in the number of crosslinked transcript copies recovered by PAR-CLIP in the presence or absence of chemically induced ER stress and the fold-change in the number of RNA-Seq reads (FPKM) in the same conditions. Genes repressed or expressed during ER stress are indicated on the X-axis. Transcripts for which there is a loss or gain of recovered crosslinked copies are indicated on the Y-axis. ( D ) Representative quantitative real-time PCR measurements of the levels of select PAR-CLIP hit mRNAs in response to ER stress in different cell types. ER stress was induced with 300 nM of the ER calcium reuptake inhibitor thapsigargin, and the cells were pre-treated for 30 min with 5 μg/ml actinomycin D to block transcription. The IRE1 specific inhibitor 4μ8C (50 μM) was used as a control for specificity. Metrics for spliced XBP1 mRNA or the canonical RIDD substrate mRNA, BLOC1S1 , were used as positive controls. Data: mean of triplicates. Error bars: SDs. ( E ) Changes in the transcript levels of select PAR-CLIP targets during ER stress measured by RNA-seq. Data: Mean FPKM values of biological duplicates. Error bars: 95% CI.
Figure Legend Snippet: Expression profiles of IRE1-associated RNAs identified by PAR-CLIP. ( A ) Left panels: Scatter plots showing the correlation between the number of crosslinked transcript copies (per million reads) recovered by PAR-CLIP and the expression level as measured by RNA-Seq in the HEK293Trex cell line that force-expresses epitope-tagged IRE1, and under the same ER stress conditions (5 μg/ml tunicamycin). The mean expression level for each transcript (obtained from biological duplicates) was normalized to the median expression level of the entire dataset, which consisted of all transcripts for which we recovered RNA-Seq reads (FPKM > 0) in a particular condition. Right panels: Histograms showing the gene expression distribution in RNA-Seq experiments. Deviations from the central tendency are color-coded, with warm colors closer to the central tendency and cool colors towards the tail ends of the distribution. The same color scheme is applied to the data points in the left panels. The dashed vertical lines indicate the standard deviation (SD) of the median-normalized data. ( B ) Waterfall plots showing the expression change in select transcripts identified by PAR-CLIP measured by RNA-Seq in the HEK293Trex cell line that force-expresses epitope-tagged IRE1, and under the same ER stress conditions (5 μg/ml tunicamycin). Bars: mean values of biological duplicates. ( C ) Scatter plots showing the fold-change in the number of crosslinked transcript copies recovered by PAR-CLIP in the presence or absence of chemically induced ER stress and the fold-change in the number of RNA-Seq reads (FPKM) in the same conditions. Genes repressed or expressed during ER stress are indicated on the X-axis. Transcripts for which there is a loss or gain of recovered crosslinked copies are indicated on the Y-axis. ( D ) Representative quantitative real-time PCR measurements of the levels of select PAR-CLIP hit mRNAs in response to ER stress in different cell types. ER stress was induced with 300 nM of the ER calcium reuptake inhibitor thapsigargin, and the cells were pre-treated for 30 min with 5 μg/ml actinomycin D to block transcription. The IRE1 specific inhibitor 4μ8C (50 μM) was used as a control for specificity. Metrics for spliced XBP1 mRNA or the canonical RIDD substrate mRNA, BLOC1S1 , were used as positive controls. Data: mean of triplicates. Error bars: SDs. ( E ) Changes in the transcript levels of select PAR-CLIP targets during ER stress measured by RNA-seq. Data: Mean FPKM values of biological duplicates. Error bars: 95% CI.

Techniques Used: Expressing, Cross-linking Immunoprecipitation, RNA Sequencing Assay, Standard Deviation, Real-time Polymerase Chain Reaction, Blocking Assay

A modifed PAR-CLIP protocol for identifying IRE1-associated RNAs. ( A ) Schematic of modified PAR-CLIP protocol. ( B ) Immunoblots showing the enrichment for IRE1 in subcellular fractions prepared by differential detergent extraction as performed in our PAR-CLIP experiments. ( C ) Immunoblot showing the relative enrichment for IRE1 in native and denaturing IP conditions. ( D ) Semi-quantitative RT-PCR showing the extent of XBP1 splicing in the HEK293Trex cell line where we force-express IRE1 in the presence or absence of chemically-induced ER stress. ER stress was induced by exposing the cells to tunicamycin. ( E ) Top: Autoradiogram of an SDS-PAGE gel in which the 5’-ends of IRE1-crosslinked RNAs were radiolabeled with polynucleotide kinase after immunoprecipitation of epitope-tagged IRE1. The crosslinked lysates were treated with the indicated amounts of RNase T1 for 30 min at 25°C. Bottom: Autoradiogram of a TBE-urea PAGE gel in which the RNA fragments purified from gel cutouts obtained from the gel in the top panel were resolved. ( F ) Autoradiogram of an SDS-PAGE gel of the eluates of denaturing IPs of epitope-tagged IRE1 after radiolabeling the 5’-end of crosslinked RNAs. The bracket on the left indicates the region of the gel that was excised for purification of IRE1-RNA complexes. The dashed line indicates where the lanes from a single gel were cropped.
Figure Legend Snippet: A modifed PAR-CLIP protocol for identifying IRE1-associated RNAs. ( A ) Schematic of modified PAR-CLIP protocol. ( B ) Immunoblots showing the enrichment for IRE1 in subcellular fractions prepared by differential detergent extraction as performed in our PAR-CLIP experiments. ( C ) Immunoblot showing the relative enrichment for IRE1 in native and denaturing IP conditions. ( D ) Semi-quantitative RT-PCR showing the extent of XBP1 splicing in the HEK293Trex cell line where we force-express IRE1 in the presence or absence of chemically-induced ER stress. ER stress was induced by exposing the cells to tunicamycin. ( E ) Top: Autoradiogram of an SDS-PAGE gel in which the 5’-ends of IRE1-crosslinked RNAs were radiolabeled with polynucleotide kinase after immunoprecipitation of epitope-tagged IRE1. The crosslinked lysates were treated with the indicated amounts of RNase T1 for 30 min at 25°C. Bottom: Autoradiogram of a TBE-urea PAGE gel in which the RNA fragments purified from gel cutouts obtained from the gel in the top panel were resolved. ( F ) Autoradiogram of an SDS-PAGE gel of the eluates of denaturing IPs of epitope-tagged IRE1 after radiolabeling the 5’-end of crosslinked RNAs. The bracket on the left indicates the region of the gel that was excised for purification of IRE1-RNA complexes. The dashed line indicates where the lanes from a single gel were cropped.

Techniques Used: Cross-linking Immunoprecipitation, Modification, Western Blot, Quantitative RT-PCR, SDS Page, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Purification, Radioactivity

3) Product Images from "The unfolded protein response and endoplasmic reticulum protein targeting machineries converge on the stress sensor IRE1"

Article Title: The unfolded protein response and endoplasmic reticulum protein targeting machineries converge on the stress sensor IRE1

Journal: eLife

doi: 10.7554/eLife.43036

IRE1 associates with mRNAs and ncRNAs. ( A ) Heat map showing the relative amounts of select transcripts in the common-core (intersection of Venn diagrams. Arbitrary threshold: copies ≥ 10). ( B ) Gene-functional categories enriched among the crosslinked protein-coding transcripts recovered in each experimental set-up grouped by gene-ontology (GO) terms. ( C ) Changes in the transcript levels of select PAR-CLIP targets during ER stress measured by RNA-seq. Data: Mean FPKM values of biological duplicates. Error bars: 95% CI. ( D ) TBE-urea PAGE gels showing the IRE1-dependent cleavage of select PAR-CLIP targets. The IRE1 specific inhibitor 4μ8C (10 μM) was used as a control for specificity.
Figure Legend Snippet: IRE1 associates with mRNAs and ncRNAs. ( A ) Heat map showing the relative amounts of select transcripts in the common-core (intersection of Venn diagrams. Arbitrary threshold: copies ≥ 10). ( B ) Gene-functional categories enriched among the crosslinked protein-coding transcripts recovered in each experimental set-up grouped by gene-ontology (GO) terms. ( C ) Changes in the transcript levels of select PAR-CLIP targets during ER stress measured by RNA-seq. Data: Mean FPKM values of biological duplicates. Error bars: 95% CI. ( D ) TBE-urea PAGE gels showing the IRE1-dependent cleavage of select PAR-CLIP targets. The IRE1 specific inhibitor 4μ8C (10 μM) was used as a control for specificity.

Techniques Used: Functional Assay, Cross-linking Immunoprecipitation, RNA Sequencing Assay, Polyacrylamide Gel Electrophoresis

Identification of IRE1-associated RNAs. ( A ) Experimental strategy. ( B ) Mutation plots showing T→C transitions are the most common mutations recovered by PAR-CLIP in biological duplicates. ( C ) Breakdown of RNA classes associated with IRE1 identified by PAR-CLIP in biological duplicates. ( D ) Venn diagrams showing the numbers of crosslinked transcripts recovered by PAR-CLIP in the presence or absence of chemically induced ER stress in biological duplicates. Cardinals indicate the total or non-coding (in parenthesis) number of transcripts in each group. ( E ) Scatter plot showing the abundance of PAR-CLIP recovered transcripts in the presence or absence of chemically induced ER stress (copies per million reads: geometric mean of copy number per transcript in biological duplicates). The histograms above and to the side of the scatter plot illustrate the frequency of transcripts that traverse the secretory pathway. The diagonal dashed line indicates a slope of 1. ( F ) Breakdown of mRNA regions associated with IRE1 in PAR-CLIP experiments. ‘Unknown region’ refers to transcripts associated with coding loci but that do not have a single annotated coding sequence ( i.e. , alternative splicing or alternative transcription initiation sites).
Figure Legend Snippet: Identification of IRE1-associated RNAs. ( A ) Experimental strategy. ( B ) Mutation plots showing T→C transitions are the most common mutations recovered by PAR-CLIP in biological duplicates. ( C ) Breakdown of RNA classes associated with IRE1 identified by PAR-CLIP in biological duplicates. ( D ) Venn diagrams showing the numbers of crosslinked transcripts recovered by PAR-CLIP in the presence or absence of chemically induced ER stress in biological duplicates. Cardinals indicate the total or non-coding (in parenthesis) number of transcripts in each group. ( E ) Scatter plot showing the abundance of PAR-CLIP recovered transcripts in the presence or absence of chemically induced ER stress (copies per million reads: geometric mean of copy number per transcript in biological duplicates). The histograms above and to the side of the scatter plot illustrate the frequency of transcripts that traverse the secretory pathway. The diagonal dashed line indicates a slope of 1. ( F ) Breakdown of mRNA regions associated with IRE1 in PAR-CLIP experiments. ‘Unknown region’ refers to transcripts associated with coding loci but that do not have a single annotated coding sequence ( i.e. , alternative splicing or alternative transcription initiation sites).

Techniques Used: Mutagenesis, Cross-linking Immunoprecipitation, Sequencing

Related Articles

Irradiation:

Article Title: Genome-wide RNA-binding analysis of the trypanosome U1 snRNP proteins U1C and U1-70K reveals cis/trans-spliceosomal network
Article Snippet: 5 × 108 procylic T. brucei cells were irradiated with UV-C light (3 × 300 mJ/cm2 ). .. In brief, U1C- or U1-70K RNA–protein complexes were purified by applying the first step of tandem-affinity purification (IgG Sepharose 6 Fast Flow, GE Healthcare), followed by phosphatase treatment, ligation of an RNA adapter at the 3′ ends of the RNA tags (T4 RNA ligase; Thermo Scientific) and radiolabeling using polynucleotide kinase treatment to allow visualization of covalent RNA–protein complexes.

Flow Cytometry:

Article Title: Genome-wide RNA-binding analysis of the trypanosome U1 snRNP proteins U1C and U1-70K reveals cis/trans-spliceosomal network
Article Snippet: .. In brief, U1C- or U1-70K RNA–protein complexes were purified by applying the first step of tandem-affinity purification (IgG Sepharose 6 Fast Flow, GE Healthcare), followed by phosphatase treatment, ligation of an RNA adapter at the 3′ ends of the RNA tags (T4 RNA ligase; Thermo Scientific) and radiolabeling using polynucleotide kinase treatment to allow visualization of covalent RNA–protein complexes. .. By tobacco-etch-virus (TEV) protease bound material was released from the beads, followed by the second affinity step (anti-protein C immunoaffinity purification).

Radioactivity:

Article Title: Genome-wide RNA-binding analysis of the trypanosome U1 snRNP proteins U1C and U1-70K reveals cis/trans-spliceosomal network
Article Snippet: .. In brief, U1C- or U1-70K RNA–protein complexes were purified by applying the first step of tandem-affinity purification (IgG Sepharose 6 Fast Flow, GE Healthcare), followed by phosphatase treatment, ligation of an RNA adapter at the 3′ ends of the RNA tags (T4 RNA ligase; Thermo Scientific) and radiolabeling using polynucleotide kinase treatment to allow visualization of covalent RNA–protein complexes. .. By tobacco-etch-virus (TEV) protease bound material was released from the beads, followed by the second affinity step (anti-protein C immunoaffinity purification).

Nucleic Acid Electrophoresis:

Article Title: Genome-wide RNA-binding analysis of the trypanosome U1 snRNP proteins U1C and U1-70K reveals cis/trans-spliceosomal network
Article Snippet: In brief, U1C- or U1-70K RNA–protein complexes were purified by applying the first step of tandem-affinity purification (IgG Sepharose 6 Fast Flow, GE Healthcare), followed by phosphatase treatment, ligation of an RNA adapter at the 3′ ends of the RNA tags (T4 RNA ligase; Thermo Scientific) and radiolabeling using polynucleotide kinase treatment to allow visualization of covalent RNA–protein complexes. .. Purified RNA–protein complexes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electro-blotting.

Ligation:

Article Title: Genome-wide RNA-binding analysis of the trypanosome U1 snRNP proteins U1C and U1-70K reveals cis/trans-spliceosomal network
Article Snippet: .. In brief, U1C- or U1-70K RNA–protein complexes were purified by applying the first step of tandem-affinity purification (IgG Sepharose 6 Fast Flow, GE Healthcare), followed by phosphatase treatment, ligation of an RNA adapter at the 3′ ends of the RNA tags (T4 RNA ligase; Thermo Scientific) and radiolabeling using polynucleotide kinase treatment to allow visualization of covalent RNA–protein complexes. .. By tobacco-etch-virus (TEV) protease bound material was released from the beads, followed by the second affinity step (anti-protein C immunoaffinity purification).

Centrifugation:

Article Title: Genome-wide RNA-binding analysis of the trypanosome U1 snRNP proteins U1C and U1-70K reveals cis/trans-spliceosomal network
Article Snippet: Extracts were cleared by centrifugation at 14 000 rpm for 30 min and subsequently, 1 ml of cleared extract was subjected to combined DNase treatment (TURBO™ DNase, Ambion, at a final concentration of 4 U/ml), and limited RNase digestion (RNase I, Ambion, at a final concentration of 0.01 U/ml), for 3 min at 37°C. .. In brief, U1C- or U1-70K RNA–protein complexes were purified by applying the first step of tandem-affinity purification (IgG Sepharose 6 Fast Flow, GE Healthcare), followed by phosphatase treatment, ligation of an RNA adapter at the 3′ ends of the RNA tags (T4 RNA ligase; Thermo Scientific) and radiolabeling using polynucleotide kinase treatment to allow visualization of covalent RNA–protein complexes.

Article Title: The unfolded protein response and endoplasmic reticulum protein targeting machineries converge on the stress sensor IRE1
Article Snippet: The purified crosslinked RNA tags were precipitated with 300 mM NaOAc pH 5.5, 1 volume of ice-cold isopropanol and 10 μg Glycoblue (Thermo Fisher Scientific) overnight at -80°C. .. RNA pellets were recovered after centrifugation at 21,000 × g for 30 min at 4°C in a tabletop microcentrifuge.

Introduce:

Article Title: Genome-wide RNA-binding analysis of the trypanosome U1 snRNP proteins U1C and U1-70K reveals cis/trans-spliceosomal network
Article Snippet: In brief, U1C- or U1-70K RNA–protein complexes were purified by applying the first step of tandem-affinity purification (IgG Sepharose 6 Fast Flow, GE Healthcare), followed by phosphatase treatment, ligation of an RNA adapter at the 3′ ends of the RNA tags (T4 RNA ligase; Thermo Scientific) and radiolabeling using polynucleotide kinase treatment to allow visualization of covalent RNA–protein complexes. .. Complexes were then recovered by proteinase K treatment. cDNA was generated by reverse transcription (Superscript III; Life Technologies), using oligonucleotides, which introduce a 5′-barcode as well as a BamHI restriction site. cDNAs obtained were size-fractionated by denaturing polyacrylamide gel electrophoresis, circularized (Circligase II, Epicentre), annealed to an oligonucleotide complementary to the BamHI restriction site, and cut between the two adapter regions by BamHI.

Purification:

Article Title: Genome-wide RNA-binding analysis of the trypanosome U1 snRNP proteins U1C and U1-70K reveals cis/trans-spliceosomal network
Article Snippet: .. In brief, U1C- or U1-70K RNA–protein complexes were purified by applying the first step of tandem-affinity purification (IgG Sepharose 6 Fast Flow, GE Healthcare), followed by phosphatase treatment, ligation of an RNA adapter at the 3′ ends of the RNA tags (T4 RNA ligase; Thermo Scientific) and radiolabeling using polynucleotide kinase treatment to allow visualization of covalent RNA–protein complexes. .. By tobacco-etch-virus (TEV) protease bound material was released from the beads, followed by the second affinity step (anti-protein C immunoaffinity purification).

Article Title: The unfolded protein response and endoplasmic reticulum protein targeting machineries converge on the stress sensor IRE1
Article Snippet: .. The purified crosslinked RNA tags were precipitated with 300 mM NaOAc pH 5.5, 1 volume of ice-cold isopropanol and 10 μg Glycoblue (Thermo Fisher Scientific) overnight at -80°C. .. RNA pellets were recovered after centrifugation at 21,000 × g for 30 min at 4°C in a tabletop microcentrifuge.

Electrophoresis:

Article Title: The unfolded protein response and endoplasmic reticulum protein targeting machineries converge on the stress sensor IRE1
Article Snippet: The dialyzers were placed immersed on a horizontal electrophoresis apparatus filled with 1× MOPS SDS buffer, and a constant voltage of 100V was applied for 2.5 hr to electroelute the crosslinked protein-RNA complexes. .. The purified crosslinked RNA tags were precipitated with 300 mM NaOAc pH 5.5, 1 volume of ice-cold isopropanol and 10 μg Glycoblue (Thermo Fisher Scientific) overnight at -80°C.

Concentration Assay:

Article Title: Genome-wide RNA-binding analysis of the trypanosome U1 snRNP proteins U1C and U1-70K reveals cis/trans-spliceosomal network
Article Snippet: Extracts were cleared by centrifugation at 14 000 rpm for 30 min and subsequently, 1 ml of cleared extract was subjected to combined DNase treatment (TURBO™ DNase, Ambion, at a final concentration of 4 U/ml), and limited RNase digestion (RNase I, Ambion, at a final concentration of 0.01 U/ml), for 3 min at 37°C. .. In brief, U1C- or U1-70K RNA–protein complexes were purified by applying the first step of tandem-affinity purification (IgG Sepharose 6 Fast Flow, GE Healthcare), followed by phosphatase treatment, ligation of an RNA adapter at the 3′ ends of the RNA tags (T4 RNA ligase; Thermo Scientific) and radiolabeling using polynucleotide kinase treatment to allow visualization of covalent RNA–protein complexes.

Article Title: The unfolded protein response and endoplasmic reticulum protein targeting machineries converge on the stress sensor IRE1
Article Snippet: The electroeluates were transferred to clean RNase-free non-stick microcentrifuge tubes and 1 volume of 2× proteinase K buffer (100 mM Tris pH 7.5, 100 mM NaCl, 20 mM EDTA, 2% SDS) and RNA-grade proteinase K (Thermo Fisher Scientific) to a final concentration of 1.2 mg/mL, were added to digest the protein by incubation for 30 min at 55°C. .. The purified crosslinked RNA tags were precipitated with 300 mM NaOAc pH 5.5, 1 volume of ice-cold isopropanol and 10 μg Glycoblue (Thermo Fisher Scientific) overnight at -80°C.

Incubation:

Article Title: The unfolded protein response and endoplasmic reticulum protein targeting machineries converge on the stress sensor IRE1
Article Snippet: The electroeluates were transferred to clean RNase-free non-stick microcentrifuge tubes and 1 volume of 2× proteinase K buffer (100 mM Tris pH 7.5, 100 mM NaCl, 20 mM EDTA, 2% SDS) and RNA-grade proteinase K (Thermo Fisher Scientific) to a final concentration of 1.2 mg/mL, were added to digest the protein by incubation for 30 min at 55°C. .. The purified crosslinked RNA tags were precipitated with 300 mM NaOAc pH 5.5, 1 volume of ice-cold isopropanol and 10 μg Glycoblue (Thermo Fisher Scientific) overnight at -80°C.

Polyacrylamide Gel Electrophoresis:

Article Title: Genome-wide RNA-binding analysis of the trypanosome U1 snRNP proteins U1C and U1-70K reveals cis/trans-spliceosomal network
Article Snippet: In brief, U1C- or U1-70K RNA–protein complexes were purified by applying the first step of tandem-affinity purification (IgG Sepharose 6 Fast Flow, GE Healthcare), followed by phosphatase treatment, ligation of an RNA adapter at the 3′ ends of the RNA tags (T4 RNA ligase; Thermo Scientific) and radiolabeling using polynucleotide kinase treatment to allow visualization of covalent RNA–protein complexes. .. Complexes were then recovered by proteinase K treatment. cDNA was generated by reverse transcription (Superscript III; Life Technologies), using oligonucleotides, which introduce a 5′-barcode as well as a BamHI restriction site. cDNAs obtained were size-fractionated by denaturing polyacrylamide gel electrophoresis, circularized (Circligase II, Epicentre), annealed to an oligonucleotide complementary to the BamHI restriction site, and cut between the two adapter regions by BamHI.

Sonication:

Article Title: Genome-wide RNA-binding analysis of the trypanosome U1 snRNP proteins U1C and U1-70K reveals cis/trans-spliceosomal network
Article Snippet: Lysates were prepared in 4 ml extraction buffer (500 mM KCl, 20 mM Tris–Cl, pH 7.7, 3 mM MgCl2 , 0.5 mM DTT) using a Dounce homogenizer (25 strokes with a type B pestle) followed by sonication. .. In brief, U1C- or U1-70K RNA–protein complexes were purified by applying the first step of tandem-affinity purification (IgG Sepharose 6 Fast Flow, GE Healthcare), followed by phosphatase treatment, ligation of an RNA adapter at the 3′ ends of the RNA tags (T4 RNA ligase; Thermo Scientific) and radiolabeling using polynucleotide kinase treatment to allow visualization of covalent RNA–protein complexes.

Western Blot:

Article Title: Genome-wide RNA-binding analysis of the trypanosome U1 snRNP proteins U1C and U1-70K reveals cis/trans-spliceosomal network
Article Snippet: In brief, U1C- or U1-70K RNA–protein complexes were purified by applying the first step of tandem-affinity purification (IgG Sepharose 6 Fast Flow, GE Healthcare), followed by phosphatase treatment, ligation of an RNA adapter at the 3′ ends of the RNA tags (T4 RNA ligase; Thermo Scientific) and radiolabeling using polynucleotide kinase treatment to allow visualization of covalent RNA–protein complexes. .. Purified RNA–protein complexes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electro-blotting.

Immunoaffinity Purification:

Article Title: Genome-wide RNA-binding analysis of the trypanosome U1 snRNP proteins U1C and U1-70K reveals cis/trans-spliceosomal network
Article Snippet: In brief, U1C- or U1-70K RNA–protein complexes were purified by applying the first step of tandem-affinity purification (IgG Sepharose 6 Fast Flow, GE Healthcare), followed by phosphatase treatment, ligation of an RNA adapter at the 3′ ends of the RNA tags (T4 RNA ligase; Thermo Scientific) and radiolabeling using polynucleotide kinase treatment to allow visualization of covalent RNA–protein complexes. .. By tobacco-etch-virus (TEV) protease bound material was released from the beads, followed by the second affinity step (anti-protein C immunoaffinity purification).

SDS Page:

Article Title: Genome-wide RNA-binding analysis of the trypanosome U1 snRNP proteins U1C and U1-70K reveals cis/trans-spliceosomal network
Article Snippet: In brief, U1C- or U1-70K RNA–protein complexes were purified by applying the first step of tandem-affinity purification (IgG Sepharose 6 Fast Flow, GE Healthcare), followed by phosphatase treatment, ligation of an RNA adapter at the 3′ ends of the RNA tags (T4 RNA ligase; Thermo Scientific) and radiolabeling using polynucleotide kinase treatment to allow visualization of covalent RNA–protein complexes. .. Purified RNA–protein complexes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electro-blotting.

Molecular Weight:

Article Title: The unfolded protein response and endoplasmic reticulum protein targeting machineries converge on the stress sensor IRE1
Article Snippet: The radioactive bands corresponding the approximate molecular weight of IRE1 (~130 kDa) and the diffuse radioactive band above it were excised from the gels, and the gel slices were placed in a water-pre-equilibrated D-tube dialyzer (EMD Millipore) with a molecular weight cut-off of 3.5 kDa. .. The purified crosslinked RNA tags were precipitated with 300 mM NaOAc pH 5.5, 1 volume of ice-cold isopropanol and 10 μg Glycoblue (Thermo Fisher Scientific) overnight at -80°C.

Generated:

Article Title: Genome-wide RNA-binding analysis of the trypanosome U1 snRNP proteins U1C and U1-70K reveals cis/trans-spliceosomal network
Article Snippet: In brief, U1C- or U1-70K RNA–protein complexes were purified by applying the first step of tandem-affinity purification (IgG Sepharose 6 Fast Flow, GE Healthcare), followed by phosphatase treatment, ligation of an RNA adapter at the 3′ ends of the RNA tags (T4 RNA ligase; Thermo Scientific) and radiolabeling using polynucleotide kinase treatment to allow visualization of covalent RNA–protein complexes. .. Complexes were then recovered by proteinase K treatment. cDNA was generated by reverse transcription (Superscript III; Life Technologies), using oligonucleotides, which introduce a 5′-barcode as well as a BamHI restriction site. cDNAs obtained were size-fractionated by denaturing polyacrylamide gel electrophoresis, circularized (Circligase II, Epicentre), annealed to an oligonucleotide complementary to the BamHI restriction site, and cut between the two adapter regions by BamHI.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher fish tag rna kit suggested hybridization protocol
    Single-cell <t>RNA-seq</t> identifies cell-specific markers. a Dot plot of top markers for all populations. Red arrows point at previously unreported eye cell-type markers. Green arrows indicate genes previously studied in the eye that were not assigned to a specific cell type. The genes without arrows are known markers used to assign cell populations. b LacZ enhancer traps for posterior candidate markers neuromusculin ( nrm ), sallimus ( sls , also known as kettin ) and photoreceptor marker couch potato ( cpo ). c Dot plots, top panel shows interommatidial populations INT and SMW, showing expression of cell-cycle genes exclusively in SMW subpopulation. Bottom panel shows glial cell populations PG and WG+SPG showing expression of cell-cycle genes exclusively in PG. The scale bar is 50 µm. d Wrapping glia markers CG9336 and NK7.1 of and their expression as revealed by <t>FISH</t> ( CG9336 ) and a GFP reporter for NK7.1 (green). Repo (red) is a glial marker. Merge image marks cells co-expressing NK7.1 and Repo, also shown with yellow arrow. Markers of PG, cpo and pigs are revealed by LacZ enhancer trap and GFP reporter, respectively. SPG are indicated by a yellow arrowhead, overlap of Repo and pigs shown with yellow arrow. The scale bar is 50 µm. The position of the MF is shown by white arrowhead
    Fish Tag Rna Kit Suggested Hybridization Protocol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fish tag rna kit suggested hybridization protocol/product/Thermo Fisher
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    fish tag rna kit suggested hybridization protocol - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    94
    Thermo Fisher double nuclear localization signal nls tagged cas9 rna
    Sequences of two <t>Cas9-induced</t> frameshift mutations (alleles pcnt tup2 and pcnt tup5 ) in the zebrafish pcnt gene. The wild-type reference sequence is on the top. The guide <t>RNA</t> targets the exon 2 of the pcnt transcript (encoded by ENSDARG00000033012). The target site is underlined and the protospacer-adjacent motif (PAM) is in orange (on the reverse strand). Insertions and deletions (indels) are indicated by blue lowercase letters and dashes, respectively. The net change of each indel mutation is noted at the right of each sequence (+, insertion; −, deletion).
    Double Nuclear Localization Signal Nls Tagged Cas9 Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/double nuclear localization signal nls tagged cas9 rna/product/Thermo Fisher
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    double nuclear localization signal nls tagged cas9 rna - by Bioz Stars, 2020-04
    94/100 stars
      Buy from Supplier

    93
    Thermo Fisher icres one strep tag ost rnas
    Effect of AUD mutations on CHIKV protein expression and RNA synthesis. (A) C2C12 cells were electroporated with <t>ICRES-RNAs</t> (wildtype (WT) and indicated AUD mutants) and cell lysates were collected at 36 h.p.e. Expression of nsP3 and capsid was analysed by western blot. Representative western blots are presented. For quantification using a LiCor Odyssey Sa fluorescence imager western blots from three independent experiments were analysed and the graph on the right shows the ratio of capsid to nsP3 expression. (B) C2C12 cells were electroporated with the indicated ICRES RNAs, cellular RNA synthesis was inhibited by actinomycin D and nascent viral RNAs were labelled with [ 3 H]-uridine. A representative autoradiograph is presented. The graph on the right shows the ratio of gRNA to sgRNA determined by quantification of three independent experiments. (C) The same RNAs were fractionated on a sucrose gradient and [ 3 H]-labelled RNAs were detected by scintillation counting of individual fractions.
    Icres One Strep Tag Ost Rnas, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/icres one strep tag ost rnas/product/Thermo Fisher
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    icres one strep tag ost rnas - by Bioz Stars, 2020-04
    93/100 stars
      Buy from Supplier

    Image Search Results


    Single-cell RNA-seq identifies cell-specific markers. a Dot plot of top markers for all populations. Red arrows point at previously unreported eye cell-type markers. Green arrows indicate genes previously studied in the eye that were not assigned to a specific cell type. The genes without arrows are known markers used to assign cell populations. b LacZ enhancer traps for posterior candidate markers neuromusculin ( nrm ), sallimus ( sls , also known as kettin ) and photoreceptor marker couch potato ( cpo ). c Dot plots, top panel shows interommatidial populations INT and SMW, showing expression of cell-cycle genes exclusively in SMW subpopulation. Bottom panel shows glial cell populations PG and WG+SPG showing expression of cell-cycle genes exclusively in PG. The scale bar is 50 µm. d Wrapping glia markers CG9336 and NK7.1 of and their expression as revealed by FISH ( CG9336 ) and a GFP reporter for NK7.1 (green). Repo (red) is a glial marker. Merge image marks cells co-expressing NK7.1 and Repo, also shown with yellow arrow. Markers of PG, cpo and pigs are revealed by LacZ enhancer trap and GFP reporter, respectively. SPG are indicated by a yellow arrowhead, overlap of Repo and pigs shown with yellow arrow. The scale bar is 50 µm. The position of the MF is shown by white arrowhead

    Journal: Nature Communications

    Article Title: Single cell RNA-sequencing identifies a metabolic aspect of apoptosis in Rbf mutant

    doi: 10.1038/s41467-018-07540-z

    Figure Lengend Snippet: Single-cell RNA-seq identifies cell-specific markers. a Dot plot of top markers for all populations. Red arrows point at previously unreported eye cell-type markers. Green arrows indicate genes previously studied in the eye that were not assigned to a specific cell type. The genes without arrows are known markers used to assign cell populations. b LacZ enhancer traps for posterior candidate markers neuromusculin ( nrm ), sallimus ( sls , also known as kettin ) and photoreceptor marker couch potato ( cpo ). c Dot plots, top panel shows interommatidial populations INT and SMW, showing expression of cell-cycle genes exclusively in SMW subpopulation. Bottom panel shows glial cell populations PG and WG+SPG showing expression of cell-cycle genes exclusively in PG. The scale bar is 50 µm. d Wrapping glia markers CG9336 and NK7.1 of and their expression as revealed by FISH ( CG9336 ) and a GFP reporter for NK7.1 (green). Repo (red) is a glial marker. Merge image marks cells co-expressing NK7.1 and Repo, also shown with yellow arrow. Markers of PG, cpo and pigs are revealed by LacZ enhancer trap and GFP reporter, respectively. SPG are indicated by a yellow arrowhead, overlap of Repo and pigs shown with yellow arrow. The scale bar is 50 µm. The position of the MF is shown by white arrowhead

    Article Snippet: Digoxigenin (DIG)-labeled RNA probes were denatured at 90 °C for 4 min, using 3–5 μL probe in 25 μL hybridization solution and hybridization was carried out following ThermoFisher FISH Tag RNA Kit suggested hybridization protocol ( https://tools.thermofisher.com/content/sfs/manuals/mp32952.pdf ).

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

    Timing of Hox gene derepression in Ph del and Pc XT109 embryos. a – d RNA FISH images illustrating the earliest ectopic Hox gene expression observed in ph del ( a , c ) and Pc XT109 ( b , d ) embryos. These images are maximum intensity projections of confocal images and dashed lines indicate where two images were merged. Arrowheads indicate few cells showing Hox gene transcription outside of their domains of expression ( Ubx : red in a , b ; abdA : green in b ; AbdB : blue in b ; Antp : green in c , d and Scr: blue in d ). Scale bars, 10 µm. e Embryos were grouped into four classes based on their developmental stage, which depended on the duration of their development after fertilization at 25 °C. f – k , Relative densities of RNA FISH spots corresponding to Ubx ( f ), abdA ( g ), AbdB ( h ), pb ( i ), Scr ( j ), and Antp ( k ) expression measured in WT, Ph del and Pc XT109 embryos during development. For simplicity, PSs wherein Hox genes of BX-C ( f – h ) and ANT-C ( i – k ) behave similarly were grouped (complete data are shown in Supplementary Figure 2 ). Red columns indicate where and when Hox gene transcription was significantly ectopically expressed in the mutants compared to WT embryos, whereas the blue columns show significant (Mann–Whitney U -test, two-tailed, P

    Journal: Nature Communications

    Article Title: Loss of PRC1 induces higher-order opening of Hox loci independently of transcription during Drosophila embryogenesis

    doi: 10.1038/s41467-018-05945-4

    Figure Lengend Snippet: Timing of Hox gene derepression in Ph del and Pc XT109 embryos. a – d RNA FISH images illustrating the earliest ectopic Hox gene expression observed in ph del ( a , c ) and Pc XT109 ( b , d ) embryos. These images are maximum intensity projections of confocal images and dashed lines indicate where two images were merged. Arrowheads indicate few cells showing Hox gene transcription outside of their domains of expression ( Ubx : red in a , b ; abdA : green in b ; AbdB : blue in b ; Antp : green in c , d and Scr: blue in d ). Scale bars, 10 µm. e Embryos were grouped into four classes based on their developmental stage, which depended on the duration of their development after fertilization at 25 °C. f – k , Relative densities of RNA FISH spots corresponding to Ubx ( f ), abdA ( g ), AbdB ( h ), pb ( i ), Scr ( j ), and Antp ( k ) expression measured in WT, Ph del and Pc XT109 embryos during development. For simplicity, PSs wherein Hox genes of BX-C ( f – h ) and ANT-C ( i – k ) behave similarly were grouped (complete data are shown in Supplementary Figure 2 ). Red columns indicate where and when Hox gene transcription was significantly ectopically expressed in the mutants compared to WT embryos, whereas the blue columns show significant (Mann–Whitney U -test, two-tailed, P

    Article Snippet: Probes were prepared using a direct labeling approach with a FISH Tag RNA Kit (Thermo Fisher Scientific, F32956), and primers used to synthesize RNA probes are listed in Supplementary Table .

    Techniques: Fluorescence In Situ Hybridization, Expressing, MANN-WHITNEY, Two Tailed Test

    Hox gene expression correlates with Hox cluster chromatin opening. a Schematic representation of ANT-C and BX-C (Hox genes in blue, positions of DNA FISH probes in red, and positions of RNA FISH probes in green; the arrows show promoters). b Schematic diagram depicting the PSs of Drosophila embryos in which measurements were performed. c Relative density of RNA FISH spots measured for each Hox gene along the anteroposterior axis of WT Drosophila embryos 3:50–7:20 after fertilization. The density of RNA FISH spots is calculated by dividing the number of spots by the area of the PS. d , e Distances between the promoters of Ubx , abdA , and AbdB (d) or lab , Scr , and Antp (e) measured within cell nuclei of WT Drosophila embryos 3:50–7:20 after fertilization along the anteroposterior axis. We calculated distances between centroids of DNA FISH spots in three dimensions. For each PS of one embryo, we computed the median distances Ubx – abdA , abdA – AbdB , Ubx – AbdB or lab – Scr , Scr – Antp , lab – Antp . Curves represent the average median distances measured in several embryos and the corresponding error bar (SEM; N ≥ 16). f , g Heat maps showing correlations between Hox gene transcription and chromatin opening in the BX-C ( f ) and the ANT-C ( g ). The physical distance (Dist) between two loci within a Hox complex measured the chromatin opening and Hox gene expression (Exp) is calculated by adding the relative density of RNA FISH spot of Hox genes comprised between these two loci. All measurements were normalized between their minimum and their maximum. r indicates correlation coefficients. h , i Distances between promoters of Ubx , abdA , and AbdB (h) or lab , Scr and Antp (i) and the closest Polycomb foci were measured in WT Drosophila embryos 3:50–7:20 after fertilization. The percentage of distances measuring less than 400 nm was calculated for each PS of one embryo. Curves represent the mean percentage measured in several embryos. The highlighted PSs show regions wherein Hox genes were found to be significantly further away from Pc foci than in Head-PS0 ( t -test, one-tailed, P

    Journal: Nature Communications

    Article Title: Loss of PRC1 induces higher-order opening of Hox loci independently of transcription during Drosophila embryogenesis

    doi: 10.1038/s41467-018-05945-4

    Figure Lengend Snippet: Hox gene expression correlates with Hox cluster chromatin opening. a Schematic representation of ANT-C and BX-C (Hox genes in blue, positions of DNA FISH probes in red, and positions of RNA FISH probes in green; the arrows show promoters). b Schematic diagram depicting the PSs of Drosophila embryos in which measurements were performed. c Relative density of RNA FISH spots measured for each Hox gene along the anteroposterior axis of WT Drosophila embryos 3:50–7:20 after fertilization. The density of RNA FISH spots is calculated by dividing the number of spots by the area of the PS. d , e Distances between the promoters of Ubx , abdA , and AbdB (d) or lab , Scr , and Antp (e) measured within cell nuclei of WT Drosophila embryos 3:50–7:20 after fertilization along the anteroposterior axis. We calculated distances between centroids of DNA FISH spots in three dimensions. For each PS of one embryo, we computed the median distances Ubx – abdA , abdA – AbdB , Ubx – AbdB or lab – Scr , Scr – Antp , lab – Antp . Curves represent the average median distances measured in several embryos and the corresponding error bar (SEM; N ≥ 16). f , g Heat maps showing correlations between Hox gene transcription and chromatin opening in the BX-C ( f ) and the ANT-C ( g ). The physical distance (Dist) between two loci within a Hox complex measured the chromatin opening and Hox gene expression (Exp) is calculated by adding the relative density of RNA FISH spot of Hox genes comprised between these two loci. All measurements were normalized between their minimum and their maximum. r indicates correlation coefficients. h , i Distances between promoters of Ubx , abdA , and AbdB (h) or lab , Scr and Antp (i) and the closest Polycomb foci were measured in WT Drosophila embryos 3:50–7:20 after fertilization. The percentage of distances measuring less than 400 nm was calculated for each PS of one embryo. Curves represent the mean percentage measured in several embryos. The highlighted PSs show regions wherein Hox genes were found to be significantly further away from Pc foci than in Head-PS0 ( t -test, one-tailed, P

    Article Snippet: Probes were prepared using a direct labeling approach with a FISH Tag RNA Kit (Thermo Fisher Scientific, F32956), and primers used to synthesize RNA probes are listed in Supplementary Table .

    Techniques: Expressing, Fluorescence In Situ Hybridization, One-tailed Test

    Sequences of two Cas9-induced frameshift mutations (alleles pcnt tup2 and pcnt tup5 ) in the zebrafish pcnt gene. The wild-type reference sequence is on the top. The guide RNA targets the exon 2 of the pcnt transcript (encoded by ENSDARG00000033012). The target site is underlined and the protospacer-adjacent motif (PAM) is in orange (on the reverse strand). Insertions and deletions (indels) are indicated by blue lowercase letters and dashes, respectively. The net change of each indel mutation is noted at the right of each sequence (+, insertion; −, deletion).

    Journal: eLife

    Article Title: Co-translational protein targeting facilitates centrosomal recruitment of PCNT during centrosome maturation in vertebrates

    doi: 10.7554/eLife.34959

    Figure Lengend Snippet: Sequences of two Cas9-induced frameshift mutations (alleles pcnt tup2 and pcnt tup5 ) in the zebrafish pcnt gene. The wild-type reference sequence is on the top. The guide RNA targets the exon 2 of the pcnt transcript (encoded by ENSDARG00000033012). The target site is underlined and the protospacer-adjacent motif (PAM) is in orange (on the reverse strand). Insertions and deletions (indels) are indicated by blue lowercase letters and dashes, respectively. The net change of each indel mutation is noted at the right of each sequence (+, insertion; −, deletion).

    Article Snippet: Capped, zebrafish codon-optimized, double nuclear localization signal (nls)-tagged Cas9 RNA, nls-zCas9-nls , was synthesized by in vitro transcription using the mMESSAGE mMACHINE T3 kit (AM1348, Thermo Fisher Scientific) with XbaI-linearized pT3TS-nls-zCas9-nls plasmid as the template.

    Techniques: Sequencing, Mutagenesis

    Effect of AUD mutations on CHIKV protein expression and RNA synthesis. (A) C2C12 cells were electroporated with ICRES-RNAs (wildtype (WT) and indicated AUD mutants) and cell lysates were collected at 36 h.p.e. Expression of nsP3 and capsid was analysed by western blot. Representative western blots are presented. For quantification using a LiCor Odyssey Sa fluorescence imager western blots from three independent experiments were analysed and the graph on the right shows the ratio of capsid to nsP3 expression. (B) C2C12 cells were electroporated with the indicated ICRES RNAs, cellular RNA synthesis was inhibited by actinomycin D and nascent viral RNAs were labelled with [ 3 H]-uridine. A representative autoradiograph is presented. The graph on the right shows the ratio of gRNA to sgRNA determined by quantification of three independent experiments. (C) The same RNAs were fractionated on a sucrose gradient and [ 3 H]-labelled RNAs were detected by scintillation counting of individual fractions.

    Journal: PLoS Pathogens

    Article Title: Multiple roles of the non-structural protein 3 (nsP3) alphavirus unique domain (AUD) during Chikungunya virus genome replication and transcription

    doi: 10.1371/journal.ppat.1007239

    Figure Lengend Snippet: Effect of AUD mutations on CHIKV protein expression and RNA synthesis. (A) C2C12 cells were electroporated with ICRES-RNAs (wildtype (WT) and indicated AUD mutants) and cell lysates were collected at 36 h.p.e. Expression of nsP3 and capsid was analysed by western blot. Representative western blots are presented. For quantification using a LiCor Odyssey Sa fluorescence imager western blots from three independent experiments were analysed and the graph on the right shows the ratio of capsid to nsP3 expression. (B) C2C12 cells were electroporated with the indicated ICRES RNAs, cellular RNA synthesis was inhibited by actinomycin D and nascent viral RNAs were labelled with [ 3 H]-uridine. A representative autoradiograph is presented. The graph on the right shows the ratio of gRNA to sgRNA determined by quantification of three independent experiments. (C) The same RNAs were fractionated on a sucrose gradient and [ 3 H]-labelled RNAs were detected by scintillation counting of individual fractions.

    Article Snippet: Precipitation of nsP3 and viral RNA Co-precipitation experiments were performed in C2C12 cells electroporated with ICRES One-Strep-tag (OST) RNAs using Streptactin-agarose (Thermo Fisher Scientific), following the manufacturers protocol.

    Techniques: Expressing, Western Blot, Fluorescence, Autoradiography

    Phenotype of AUD mutations in the production of infectious virus. (A) ICRES-RNAs, wildtype (WT) and indicated mutants, were electroporated into C2C12 cells and supernatants were collected at 48 h.p.e. Virus was titrated by plaque assay in BHK-21 cells. Significant difference denoted by ** (P

    Journal: PLoS Pathogens

    Article Title: Multiple roles of the non-structural protein 3 (nsP3) alphavirus unique domain (AUD) during Chikungunya virus genome replication and transcription

    doi: 10.1371/journal.ppat.1007239

    Figure Lengend Snippet: Phenotype of AUD mutations in the production of infectious virus. (A) ICRES-RNAs, wildtype (WT) and indicated mutants, were electroporated into C2C12 cells and supernatants were collected at 48 h.p.e. Virus was titrated by plaque assay in BHK-21 cells. Significant difference denoted by ** (P

    Article Snippet: Precipitation of nsP3 and viral RNA Co-precipitation experiments were performed in C2C12 cells electroporated with ICRES One-Strep-tag (OST) RNAs using Streptactin-agarose (Thermo Fisher Scientific), following the manufacturers protocol.

    Techniques: Plaque Assay

    CHIKV genome RNA association with nsP3 during virus replication. C2C12 cells were electroporated with ICRES nsP3-TST or untagged ICRES RNAs (wildtype (WT) and P247A/V248A). (A) Supernatants were harvested at 48 h.p.e and titrated by plaque assay on BHK-21 cells. Data are displayed as the means ± S.E. of three experimental replicates. Cell lysates were collected at 60 h.p.e. and nsP3-TST was precipitated with Streptactin-sepharose beads. Bound proteins and input lysates were subjected to western blotting (B) and both co-precipitated RNAs and lysate samples were extracted by TRIzol and quantified by qRT-PCR (C). The ratio of gRNA to nsP3 is depicted graphically (D).

    Journal: PLoS Pathogens

    Article Title: Multiple roles of the non-structural protein 3 (nsP3) alphavirus unique domain (AUD) during Chikungunya virus genome replication and transcription

    doi: 10.1371/journal.ppat.1007239

    Figure Lengend Snippet: CHIKV genome RNA association with nsP3 during virus replication. C2C12 cells were electroporated with ICRES nsP3-TST or untagged ICRES RNAs (wildtype (WT) and P247A/V248A). (A) Supernatants were harvested at 48 h.p.e and titrated by plaque assay on BHK-21 cells. Data are displayed as the means ± S.E. of three experimental replicates. Cell lysates were collected at 60 h.p.e. and nsP3-TST was precipitated with Streptactin-sepharose beads. Bound proteins and input lysates were subjected to western blotting (B) and both co-precipitated RNAs and lysate samples were extracted by TRIzol and quantified by qRT-PCR (C). The ratio of gRNA to nsP3 is depicted graphically (D).

    Article Snippet: Precipitation of nsP3 and viral RNA Co-precipitation experiments were performed in C2C12 cells electroporated with ICRES One-Strep-tag (OST) RNAs using Streptactin-agarose (Thermo Fisher Scientific), following the manufacturers protocol.

    Techniques: Plaque Assay, Western Blot, Quantitative RT-PCR

    Phenotype of AUD mutations in virus entry, release and assembly. (A) C2C12 cells were infected with CHIKV (wildtype (WT) and indicated AUD mutants) at MOI of 1. At 24 h.p.i, cells were washed with PBS and resuspended in 1 ml fresh medium. Cell suspensions were freeze/thawed 3 times to release intracellular virus. Genome RNA was quantified by qRT-PCR (blue bars), and virus titrated by plaque assay (red bars). (B) Graphical representation of the ratio of infectivity to genomic RNA. (C) Extracellular (blue bars) and intracellular (red bars) viruses were collected at 36 h.p.e from C2C12 cells electroporated with the indicated ICRES RNAs, and titrated by plaque assay. (D) Graphical representation of the ratio of extracellular to intracellular virus titres. Significant difference denoted by * (P

    Journal: PLoS Pathogens

    Article Title: Multiple roles of the non-structural protein 3 (nsP3) alphavirus unique domain (AUD) during Chikungunya virus genome replication and transcription

    doi: 10.1371/journal.ppat.1007239

    Figure Lengend Snippet: Phenotype of AUD mutations in virus entry, release and assembly. (A) C2C12 cells were infected with CHIKV (wildtype (WT) and indicated AUD mutants) at MOI of 1. At 24 h.p.i, cells were washed with PBS and resuspended in 1 ml fresh medium. Cell suspensions were freeze/thawed 3 times to release intracellular virus. Genome RNA was quantified by qRT-PCR (blue bars), and virus titrated by plaque assay (red bars). (B) Graphical representation of the ratio of infectivity to genomic RNA. (C) Extracellular (blue bars) and intracellular (red bars) viruses were collected at 36 h.p.e from C2C12 cells electroporated with the indicated ICRES RNAs, and titrated by plaque assay. (D) Graphical representation of the ratio of extracellular to intracellular virus titres. Significant difference denoted by * (P

    Article Snippet: Precipitation of nsP3 and viral RNA Co-precipitation experiments were performed in C2C12 cells electroporated with ICRES One-Strep-tag (OST) RNAs using Streptactin-agarose (Thermo Fisher Scientific), following the manufacturers protocol.

    Techniques: Infection, Quantitative RT-PCR, Plaque Assay