rna fragmentation reagents  (Thermo Fisher)


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    Name:
    RNA Fragmentation Reagents
    Description:
    For the fragmentation of aRNA prior to hybridization with oligonucleotide arrays The protocol is simple fast and can be completed in less than 20 min Sufficient Ambion reagents are included for 200 rxns Many protocols for using amplified RNA in microarray analysis recommend fragmenting the aRNA prior to hybridization with an oligonucleotide microarray This fragmentation step improves hybridization kinetics with the arrayed oligonucleotides and can lead to enhanced signal The protocol is simple Mix the aRNA with the Ambion 10X RNA Fragmentation Reagent and heat to 70°C for 15 min The rxn is then stopped by the addition of the Stop Solution and placed on ice until ready for hybridization with the microarray The average size of the resulting fragments will be 60 200 nucleotides Up to 10 µg of RNA can be fragmented in a 10 µL rxn
    Catalog Number:
    am8740
    Price:
    None
    Applications:
    RNAi, Epigenetics & Non-Coding RNA Research|miRNA & Non-Coding RNA Analysis|Microarray Hybridization & General Reagents
    Category:
    Lab Reagents and Chemicals
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    Structured Review

    Thermo Fisher rna fragmentation reagents
    Sanger-based verification of two novel methylated positions in S. <t>solfataricus</t> rRNA identified using <t>RNA-seq.</t> Bisulfite-converted sequences were amplified using amplicon-specific primers and sequenced. (A) Position C1369 in the S. solfataricus 16S rRNA. (B) Position C2643 in the S. solfataricus 23S rRNA.
    For the fragmentation of aRNA prior to hybridization with oligonucleotide arrays The protocol is simple fast and can be completed in less than 20 min Sufficient Ambion reagents are included for 200 rxns Many protocols for using amplified RNA in microarray analysis recommend fragmenting the aRNA prior to hybridization with an oligonucleotide microarray This fragmentation step improves hybridization kinetics with the arrayed oligonucleotides and can lead to enhanced signal The protocol is simple Mix the aRNA with the Ambion 10X RNA Fragmentation Reagent and heat to 70°C for 15 min The rxn is then stopped by the addition of the Stop Solution and placed on ice until ready for hybridization with the microarray The average size of the resulting fragments will be 60 200 nucleotides Up to 10 µg of RNA can be fragmented in a 10 µL rxn
    https://www.bioz.com/result/rna fragmentation reagents/product/Thermo Fisher
    Average 99 stars, based on 147 article reviews
    Price from $9.99 to $1999.99
    rna fragmentation reagents - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) Product Images from "Transcriptome-Wide Mapping of 5-methylcytidine RNA Modifications in Bacteria, Archaea, and Yeast Reveals m5C within Archaeal mRNAs"

    Article Title: Transcriptome-Wide Mapping of 5-methylcytidine RNA Modifications in Bacteria, Archaea, and Yeast Reveals m5C within Archaeal mRNAs

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003602

    Sanger-based verification of two novel methylated positions in S. solfataricus rRNA identified using RNA-seq. Bisulfite-converted sequences were amplified using amplicon-specific primers and sequenced. (A) Position C1369 in the S. solfataricus 16S rRNA. (B) Position C2643 in the S. solfataricus 23S rRNA.
    Figure Legend Snippet: Sanger-based verification of two novel methylated positions in S. solfataricus rRNA identified using RNA-seq. Bisulfite-converted sequences were amplified using amplicon-specific primers and sequenced. (A) Position C1369 in the S. solfataricus 16S rRNA. (B) Position C2643 in the S. solfataricus 23S rRNA.

    Techniques Used: Methylation, RNA Sequencing Assay, Amplification

    RNA immunoprecipitation with modification-specific antibodies. Shown is the coverage of Illumina-sequenced cDNA following RNA fragmentation, antibody pulldown, reverse transcription and sequencing. Black line, pulldown performed with an anti-5-methylcitosine (hm 5 C) antibody; green line, pulldown performed with an anti-5-hydroxy-methylcitosine antibody; red line, input RNA (no antibody applied). X-axis, position along the genome; Y-axis (right), read coverage of the sequenced anti-m 5 C library; Y-axis (left), fold enrichment of peaks related to median coverage along the gene. The coverage of the anti-hm 5 C and input libraries was normalized using the median of the anti-m 5 C library as a reference point. (A) The 23S gene of S. solfataricus . Peaks corresponding to positions 2121 and 2643 in the gene (875,473 and 875,995 relative to the S. solfataricus genome, respectively) are marked. Another peak, which did not come up in our bisulfite-based analysis, is observed around position 2760. (B) The 16S gene of S. solfataricus . A single peak corresponding to position 1369 in the gene (position 873,040 relative to the genome) is marked. (C–E) Antibody pulldown of m 5 C modifications in protein-coding genes from Table 3 .
    Figure Legend Snippet: RNA immunoprecipitation with modification-specific antibodies. Shown is the coverage of Illumina-sequenced cDNA following RNA fragmentation, antibody pulldown, reverse transcription and sequencing. Black line, pulldown performed with an anti-5-methylcitosine (hm 5 C) antibody; green line, pulldown performed with an anti-5-hydroxy-methylcitosine antibody; red line, input RNA (no antibody applied). X-axis, position along the genome; Y-axis (right), read coverage of the sequenced anti-m 5 C library; Y-axis (left), fold enrichment of peaks related to median coverage along the gene. The coverage of the anti-hm 5 C and input libraries was normalized using the median of the anti-m 5 C library as a reference point. (A) The 23S gene of S. solfataricus . Peaks corresponding to positions 2121 and 2643 in the gene (875,473 and 875,995 relative to the S. solfataricus genome, respectively) are marked. Another peak, which did not come up in our bisulfite-based analysis, is observed around position 2760. (B) The 16S gene of S. solfataricus . A single peak corresponding to position 1369 in the gene (position 873,040 relative to the genome) is marked. (C–E) Antibody pulldown of m 5 C modifications in protein-coding genes from Table 3 .

    Techniques Used: Immunoprecipitation, Modification, Sequencing

    Methylated positions in mRNAs of S. solfataricus . (A) Representative position in S. solfataricus oxidoreductase gene (locus: SSO3054 ). Colors and axes are as in Fig. 2 . (B) Sanger-based verification of the position in panel A, on bisulfite-treated reverse transcribed RNA (C) Sanger-based sequencing of the same position in panel B, on bisulfite-treated DNA, as a negative control (D) Methylated positions in mRNAs of S. solfataricus show a consensus motif. Shown are the sequences flanking the methylated positions (red) identified in mRNAs of S. solfataricus ( Table 3 ). Recurring sequence motif is underlined. (E) Visual representation of consensus sequence motif, prepared using weblogo [42] .
    Figure Legend Snippet: Methylated positions in mRNAs of S. solfataricus . (A) Representative position in S. solfataricus oxidoreductase gene (locus: SSO3054 ). Colors and axes are as in Fig. 2 . (B) Sanger-based verification of the position in panel A, on bisulfite-treated reverse transcribed RNA (C) Sanger-based sequencing of the same position in panel B, on bisulfite-treated DNA, as a negative control (D) Methylated positions in mRNAs of S. solfataricus show a consensus motif. Shown are the sequences flanking the methylated positions (red) identified in mRNAs of S. solfataricus ( Table 3 ). Recurring sequence motif is underlined. (E) Visual representation of consensus sequence motif, prepared using weblogo [42] .

    Techniques Used: Methylation, Sequencing, Negative Control

    2) Product Images from "Model Uracil-Rich RNAs and Membrane Protein mRNAs Interact Specifically with Cold Shock Proteins in Escherichia coli"

    Article Title: Model Uracil-Rich RNAs and Membrane Protein mRNAs Interact Specifically with Cold Shock Proteins in Escherichia coli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0134413

    Identification of protein-U-rich RNA interactions. Ra and Rb were synthesized in vitro with or without biotinylated CTP. The RNAs were incubated with E . coli extract (S300) in the presence of 15 mM MgCl 2 and then trapped by streptavidin beads. After extensive wash, the bound material was released in urea and SDS-containing buffer, and analyzed by (A) RNA-PAGE, stained with ethidium bromide and (B) protein Tris-tricine SDS-PAGE, stained with Instant blue. (*) Indicates specific U-rich RNA-binding proteins. These proteins were identified by mass spectrometry as CspE and CspC. (C) Biotinylated Ra-Rd were incubated with purified 6His-CspE or 6His-CspC in the presence of 15 mM Mg 2+ . RNA-protein complexes were trapped by streptavidin beads, eluted with SDS, separated by tris-tricine SDS-PAGE, and the gels were stained with Instant blue. Quantitation was performed by densitometry and expressed as percentage of the proteins that were eluted with Ra. (D) In vitro synthesized biotinylated Ra-Rd were incubated with an E . coli S300 fraction in the presence of 2 or 20 mM Mg +2 . Uper panel, Eluted material was separated by tris-tricine SDS-PAGE and stained with instant blue. Lower panel, Western blot analysis of the eluted material with anti-CspE antibodies. (E) Biotinylated Ra and Rb (0.17 μM) were incubated with increasing concentrations of purified 6His-CspC (0.17–2.2 μM). RNA-protein complexes were trapped by streptavidin beads, and after wash treated with RNaseA and eluted with SDS buffer (see methods ). Protein samples containing the same amount of RNA were separated by tris-tricine SDS-PAGE, and analyzed by Western blotting with anti CspE antibodies. (F) Quantitation of protein bands shown in E .
    Figure Legend Snippet: Identification of protein-U-rich RNA interactions. Ra and Rb were synthesized in vitro with or without biotinylated CTP. The RNAs were incubated with E . coli extract (S300) in the presence of 15 mM MgCl 2 and then trapped by streptavidin beads. After extensive wash, the bound material was released in urea and SDS-containing buffer, and analyzed by (A) RNA-PAGE, stained with ethidium bromide and (B) protein Tris-tricine SDS-PAGE, stained with Instant blue. (*) Indicates specific U-rich RNA-binding proteins. These proteins were identified by mass spectrometry as CspE and CspC. (C) Biotinylated Ra-Rd were incubated with purified 6His-CspE or 6His-CspC in the presence of 15 mM Mg 2+ . RNA-protein complexes were trapped by streptavidin beads, eluted with SDS, separated by tris-tricine SDS-PAGE, and the gels were stained with Instant blue. Quantitation was performed by densitometry and expressed as percentage of the proteins that were eluted with Ra. (D) In vitro synthesized biotinylated Ra-Rd were incubated with an E . coli S300 fraction in the presence of 2 or 20 mM Mg +2 . Uper panel, Eluted material was separated by tris-tricine SDS-PAGE and stained with instant blue. Lower panel, Western blot analysis of the eluted material with anti-CspE antibodies. (E) Biotinylated Ra and Rb (0.17 μM) were incubated with increasing concentrations of purified 6His-CspC (0.17–2.2 μM). RNA-protein complexes were trapped by streptavidin beads, and after wash treated with RNaseA and eluted with SDS buffer (see methods ). Protein samples containing the same amount of RNA were separated by tris-tricine SDS-PAGE, and analyzed by Western blotting with anti CspE antibodies. (F) Quantitation of protein bands shown in E .

    Techniques Used: Synthesized, In Vitro, Incubation, Polyacrylamide Gel Electrophoresis, Staining, SDS Page, RNA Binding Assay, Mass Spectrometry, Purification, Quantitation Assay, Western Blot

    High throughput sequencing of endogenous RNAs that co-purify with 6His-CspE. (A) Wild type E . coli expressing CspE-6His were disrupted in the presence of either 2 mM or 15 mM [Mg 2+ ] (top and bottom panels, respectively) and the total cell extracts were subjected to metal affinity chromatography using Talon resin. RNA was prepared from the total cell extracts and the imidazole-eluted material (see Fig 3 ) and subjected to high throughput sequencing. The amount of CspE-6His bound MPRs (left panels) and CPRs (right panels) is plotted as a function of the amount of the same mRNAs in the total extract. (B) CspE-binding of all detected mRNAs was calculated as [RPKM CspE-bound / RPKM extract ]. The quota of MPRs and CPRs in each 10 th percentile along the CspE-association landscape is presented as a moving average plot. (C) This panel shows the CspE-binding values in the presence of 2 mM [Mg 2+ ] for selected MPRs and CPRs that were similarly analyzed by qPCR (see Fig 3D for comparison). (D) An independent experiment that shows the CspE-binding values for selected MPRs and CPRs in the presence of 15 mM [Mg 2+ ].
    Figure Legend Snippet: High throughput sequencing of endogenous RNAs that co-purify with 6His-CspE. (A) Wild type E . coli expressing CspE-6His were disrupted in the presence of either 2 mM or 15 mM [Mg 2+ ] (top and bottom panels, respectively) and the total cell extracts were subjected to metal affinity chromatography using Talon resin. RNA was prepared from the total cell extracts and the imidazole-eluted material (see Fig 3 ) and subjected to high throughput sequencing. The amount of CspE-6His bound MPRs (left panels) and CPRs (right panels) is plotted as a function of the amount of the same mRNAs in the total extract. (B) CspE-binding of all detected mRNAs was calculated as [RPKM CspE-bound / RPKM extract ]. The quota of MPRs and CPRs in each 10 th percentile along the CspE-association landscape is presented as a moving average plot. (C) This panel shows the CspE-binding values in the presence of 2 mM [Mg 2+ ] for selected MPRs and CPRs that were similarly analyzed by qPCR (see Fig 3D for comparison). (D) An independent experiment that shows the CspE-binding values for selected MPRs and CPRs in the presence of 15 mM [Mg 2+ ].

    Techniques Used: Next-Generation Sequencing, Expressing, Affinity Chromatography, Binding Assay, Real-time Polymerase Chain Reaction

    6His-CspE/C pull-down experiments. E . coli cells co-expressing Ra, or Rb, or Rc, or Rd together with 6His-CspE or 6His-CspC were lysed, and extracts were incubated with Talon resin, for immobilizing 6His-CspE or 6His-CspC and their bound RNAs. (A) Samples from the various purification steps were analyzed by Westerm blotting with anti CspE antibodies. (B) Left panel, the eluates were treated with DNase or RNase and analyzed by Agarose gel. Right panel, the quality and size of the eluted RNA were analyzed by tapestation. (C) The total extract and the CspE/CspC bound RNAs were analyzed by qPCR with the corresponding primers to Ra, Rb, Rc, or Rd. Primers to rrl, rrs, and rnpB were utilized as controls. Values were calculated as 2 extract Ct / 2 pull-down Ct (see methods ). Error bars indicate SEM (n = 3). (D) RNA was extracted from disrupted cells and the total steady state amount of R transcripts was measured by qPCR. Endogenous RNAs ssrA and rnpB were used as controls to assure unbiased results. Since both of the controls were similarly expressed in the various samples, we chose rnpB expression as a standard for calculating the relative quantity (RQ) of each R transcripts. The RQ of Ra is defined as = 1. (E) 6His-CspE (upper panel) or 6His-CspC (lower panel) were purified from wild type E . coli extracts with Talon resin. The total extracts and the eluates were analyzed by qPCR with primers to various MPRs and CPRs and analyzed as in (C) . Error bars indicate SEM (n = 3). Right panels, an average ratio of [bound]/[total] is shown for each experiment.
    Figure Legend Snippet: 6His-CspE/C pull-down experiments. E . coli cells co-expressing Ra, or Rb, or Rc, or Rd together with 6His-CspE or 6His-CspC were lysed, and extracts were incubated with Talon resin, for immobilizing 6His-CspE or 6His-CspC and their bound RNAs. (A) Samples from the various purification steps were analyzed by Westerm blotting with anti CspE antibodies. (B) Left panel, the eluates were treated with DNase or RNase and analyzed by Agarose gel. Right panel, the quality and size of the eluted RNA were analyzed by tapestation. (C) The total extract and the CspE/CspC bound RNAs were analyzed by qPCR with the corresponding primers to Ra, Rb, Rc, or Rd. Primers to rrl, rrs, and rnpB were utilized as controls. Values were calculated as 2 extract Ct / 2 pull-down Ct (see methods ). Error bars indicate SEM (n = 3). (D) RNA was extracted from disrupted cells and the total steady state amount of R transcripts was measured by qPCR. Endogenous RNAs ssrA and rnpB were used as controls to assure unbiased results. Since both of the controls were similarly expressed in the various samples, we chose rnpB expression as a standard for calculating the relative quantity (RQ) of each R transcripts. The RQ of Ra is defined as = 1. (E) 6His-CspE (upper panel) or 6His-CspC (lower panel) were purified from wild type E . coli extracts with Talon resin. The total extracts and the eluates were analyzed by qPCR with primers to various MPRs and CPRs and analyzed as in (C) . Error bars indicate SEM (n = 3). Right panels, an average ratio of [bound]/[total] is shown for each experiment.

    Techniques Used: Expressing, Incubation, Purification, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction

    Characterization of CspE-, CspC-, and CspC/E deleted cells. (A) PCR analysis of the cspE and cspC in E . coli Δ cspE , Δ cspC , or Δ cspC Δ cspE :: kan R . (B) Western blot analysis with anti-CspE antibodies of total extracts from wild type and the deleted E . coli strains. (C) (D) In vitro synthesized biotinylated Ra or Rb were incubated with S300 fractions of wild type E . coli or E . coli Δ cspE , or E . coli Δ cspE Δ cspC in the presence of 2 mM Mg 2+ . The RNA-protein complexes were purified by streptavidin beads, eluted with SDS buffer and separated by tris-tricine SDS-PAGE. The gels were stained with Instant blue.
    Figure Legend Snippet: Characterization of CspE-, CspC-, and CspC/E deleted cells. (A) PCR analysis of the cspE and cspC in E . coli Δ cspE , Δ cspC , or Δ cspC Δ cspE :: kan R . (B) Western blot analysis with anti-CspE antibodies of total extracts from wild type and the deleted E . coli strains. (C) (D) In vitro synthesized biotinylated Ra or Rb were incubated with S300 fractions of wild type E . coli or E . coli Δ cspE , or E . coli Δ cspE Δ cspC in the presence of 2 mM Mg 2+ . The RNA-protein complexes were purified by streptavidin beads, eluted with SDS buffer and separated by tris-tricine SDS-PAGE. The gels were stained with Instant blue.

    Techniques Used: Polymerase Chain Reaction, Western Blot, In Vitro, Synthesized, Incubation, Purification, SDS Page, Staining

    3) Product Images from "Genes adapt to outsmart gene targeting strategies in mutant mouse strains by skipping exons to reinitiate transcription and translation"

    Article Title: Genes adapt to outsmart gene targeting strategies in mutant mouse strains by skipping exons to reinitiate transcription and translation

    Journal: bioRxiv

    doi: 10.1101/2020.04.22.041087

    Targeted KO-first targeting strategy in Rhbdf1 (A3) generates novel transcripts and N-terminally truncated functional proteins a. Schematic of the strategy used by Li et al. for generation of Rhbdf1 −/− homozygous mutant mice; the Rhbdf1 KO-first allele was crossed to Flp recombinase mice to remove the FRT-flanked “lacZ reporter and a neomycin resistance (neo) gene” to generate conditional-ready mice, which were later crossed with cre transgenic mice to excise the floxed gene segment (exons 4-11), generating Rhbdf1 −/− homozygous mutant mice (hereafter referred as viable2 mice, Rhbdf1 v2/v2 mice). b. Whole-exome sequencing of spleen tissue from Rhbdf1 v2/v2 mice showing loss of exons 4 through 11 in Rhbdf1 v2/v2 mutant mice. c. RT-PCR on spleens from Rhbdf1 +/+ and Rhbdf1 v2/v2 mutant mice using primers to amplify exons 6 through 8, exons 7 through 10, and exons 16 and 17. Exons 4-11 are deleted in Rhbdf1 v2/v2 mutant mice; hence no amplicons were generated using either exon 6 forward and exon 8 reverse, or exon 7 forward and exon 10 reverse, primers. However, exon 16 forward and exon 17 reverse primers generated a 211-bp product. d. RNA-Seq analysis of spleens from Rhbdf1 v2/v2 mutant mice indicating loss of exons 4 through 11; however, there is strong evidence for mutant mRNA, as indicated by the presence of the rest of the transcript, which encodes exons 12 through 18 and is not degraded by the nonsense-mediated decay mechanism. e. Schematic representation of exons and introns in the Rhbdf1 v2/v2 mutant allele. 5’ RACE using a gene-specific exon 16-17 fusion primer (GSP) was used to obtain 5’ ends of the Rhbdf1 v2/v2 mutant mRNA. We identified several novel mutant mRNAs with different translation initiation sites that could potentially generate N-terminally truncated RHBDF1 mutant proteins. See supplemental figures for variant protein and 5’ UTR sequences. Alternative exons are indicated as red boxes; predicted translation initiation sites are indicated by “START,” and termination codons are indicated by “STOP.” f. C-terminal Myc-DDK-tagged Rhbdf1 v2/v2 variant protein 1 (lanes 1, 2) or variant protein 2 (lanes 3,4), or empty vector (lanes 5, 6) were transiently expressed in 293T cells, and cell lysates were analyzed using western blotting with FLAG-specific antibody. After visualization of blots with a G:Box chemiluminescent imaging system, blots were washed, blocked in 5% nonfat dry milk, and re-probed with anti-actin antibody. g. Rescue of phenotype in Rhbdf1 −/− MEFs. Rhbdf1 +/+ (top) and Rhbdf1 −/− (bottom) MEFs were transiently transfected with 2 μg of either variant 1 or variant 2 vectors, or an empty vector, using Lipofectamine LTX. 48 h post-transfection, cells were stimulated overnight with either DMSO or 100 nM PMA, and cell-culture supernatants were analyzed using a mouse AREG ELISA kit. Data represent mean ± S.D; *p
    Figure Legend Snippet: Targeted KO-first targeting strategy in Rhbdf1 (A3) generates novel transcripts and N-terminally truncated functional proteins a. Schematic of the strategy used by Li et al. for generation of Rhbdf1 −/− homozygous mutant mice; the Rhbdf1 KO-first allele was crossed to Flp recombinase mice to remove the FRT-flanked “lacZ reporter and a neomycin resistance (neo) gene” to generate conditional-ready mice, which were later crossed with cre transgenic mice to excise the floxed gene segment (exons 4-11), generating Rhbdf1 −/− homozygous mutant mice (hereafter referred as viable2 mice, Rhbdf1 v2/v2 mice). b. Whole-exome sequencing of spleen tissue from Rhbdf1 v2/v2 mice showing loss of exons 4 through 11 in Rhbdf1 v2/v2 mutant mice. c. RT-PCR on spleens from Rhbdf1 +/+ and Rhbdf1 v2/v2 mutant mice using primers to amplify exons 6 through 8, exons 7 through 10, and exons 16 and 17. Exons 4-11 are deleted in Rhbdf1 v2/v2 mutant mice; hence no amplicons were generated using either exon 6 forward and exon 8 reverse, or exon 7 forward and exon 10 reverse, primers. However, exon 16 forward and exon 17 reverse primers generated a 211-bp product. d. RNA-Seq analysis of spleens from Rhbdf1 v2/v2 mutant mice indicating loss of exons 4 through 11; however, there is strong evidence for mutant mRNA, as indicated by the presence of the rest of the transcript, which encodes exons 12 through 18 and is not degraded by the nonsense-mediated decay mechanism. e. Schematic representation of exons and introns in the Rhbdf1 v2/v2 mutant allele. 5’ RACE using a gene-specific exon 16-17 fusion primer (GSP) was used to obtain 5’ ends of the Rhbdf1 v2/v2 mutant mRNA. We identified several novel mutant mRNAs with different translation initiation sites that could potentially generate N-terminally truncated RHBDF1 mutant proteins. See supplemental figures for variant protein and 5’ UTR sequences. Alternative exons are indicated as red boxes; predicted translation initiation sites are indicated by “START,” and termination codons are indicated by “STOP.” f. C-terminal Myc-DDK-tagged Rhbdf1 v2/v2 variant protein 1 (lanes 1, 2) or variant protein 2 (lanes 3,4), or empty vector (lanes 5, 6) were transiently expressed in 293T cells, and cell lysates were analyzed using western blotting with FLAG-specific antibody. After visualization of blots with a G:Box chemiluminescent imaging system, blots were washed, blocked in 5% nonfat dry milk, and re-probed with anti-actin antibody. g. Rescue of phenotype in Rhbdf1 −/− MEFs. Rhbdf1 +/+ (top) and Rhbdf1 −/− (bottom) MEFs were transiently transfected with 2 μg of either variant 1 or variant 2 vectors, or an empty vector, using Lipofectamine LTX. 48 h post-transfection, cells were stimulated overnight with either DMSO or 100 nM PMA, and cell-culture supernatants were analyzed using a mouse AREG ELISA kit. Data represent mean ± S.D; *p

    Techniques Used: Functional Assay, Mutagenesis, Mouse Assay, Transgenic Assay, Sequencing, Reverse Transcription Polymerase Chain Reaction, Generated, RNA Sequencing Assay, Variant Assay, Plasmid Preparation, Western Blot, Imaging, Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay

    4) Product Images from "Structural modularity of the XIST ribonucleoprotein complex"

    Article Title: Structural modularity of the XIST ribonucleoprotein complex

    Journal: bioRxiv

    doi: 10.1101/837229

    eCLIP analysis of XIST-associated proteins reveal modular domains. (A) Annotation of the human XIST RNA. XIST exons and repeats, phylogenetic conservation (PhastCons 100 and Placental PhyloP from UCSC), and PARIS data in HEK293 cells are shown, same as Figure 1D . (B) Unsupervised clustering of XIST-protein interaction profiles in 100nt windows for all 242 samples of the 121 proteins, two biological replicates each. (C) PCA analysis of all eCLIP data in 100nt windows. The mean and first 7 principal components are displayed together with percentage of variation explained by each component on the right. (D) List of proteins associated with each domain. Black and blue letters: enriched proteins based on eCLIP. Black: broad and clustered binding. Blue letters: focal binding. Red letters: enriched proteins based on fRIP-seq. (E) RBP interaction data for the A domain. The left panel shows enrichment of 6 RBPs along the entire XIST RNA based on eCLIP. The three vertical lines highlight the A-repeat region and the SRSF1 and U2AF1 peaks on exon 2. The middle panel shows zoom-in to the A-repeat domain and the vertical lines indicate the crosslinking positions of the six proteins on the repeat units. The right panel shows the average signal on a single 24nt repeat with 10nt flanking spacer sequences on each side, and the vertical lines mark the start and end of the 24nt repeat sequence. Conservation, icSHAPE and SPEN iCLIP data were from ( Lu et al., 2016 ). One replicate was shown for the eCLIP data of each protein. (F) RBP interaction data for the BCD domain. In the left panel, human 293T cell PARIS data, repeat annotations and eCLIP data are shown for the entire exon 1 ( Lu et al., 2016 ). Vertical lines mark the boundaries of the BCD domain and the internal repetitive sequences in the D-repeat. The right panel shows the average of all D repeats (290nt per unit) and the consensus HNRNP binding site. One replicate was shown for the eCLIP data of each protein. (G) RBP interaction data for the E domain. Parts of exons 1 and 6, and the entire exons 2-5 are shown, together with human 293T PARIS data ( Lu et al., 2016 ). The vertical lines mark the boundaries of the “stem” and “loop” regions of this giant stemloop. One replicate was shown for the eCLIP data of each protein. (H) Schematic RBP interaction model for the E domain. Only the four proteins that mark the “stem” and “loop” are shown. (I) Comparison of protein binding sites on XIST based on eCLIP and the sequence motifs. eCLIP data are normalized against size-matched input in 100nt windows. The density profile was calculated based on sequence motifs in 300nt windows and 50nt steps. See also Figure S2 .
    Figure Legend Snippet: eCLIP analysis of XIST-associated proteins reveal modular domains. (A) Annotation of the human XIST RNA. XIST exons and repeats, phylogenetic conservation (PhastCons 100 and Placental PhyloP from UCSC), and PARIS data in HEK293 cells are shown, same as Figure 1D . (B) Unsupervised clustering of XIST-protein interaction profiles in 100nt windows for all 242 samples of the 121 proteins, two biological replicates each. (C) PCA analysis of all eCLIP data in 100nt windows. The mean and first 7 principal components are displayed together with percentage of variation explained by each component on the right. (D) List of proteins associated with each domain. Black and blue letters: enriched proteins based on eCLIP. Black: broad and clustered binding. Blue letters: focal binding. Red letters: enriched proteins based on fRIP-seq. (E) RBP interaction data for the A domain. The left panel shows enrichment of 6 RBPs along the entire XIST RNA based on eCLIP. The three vertical lines highlight the A-repeat region and the SRSF1 and U2AF1 peaks on exon 2. The middle panel shows zoom-in to the A-repeat domain and the vertical lines indicate the crosslinking positions of the six proteins on the repeat units. The right panel shows the average signal on a single 24nt repeat with 10nt flanking spacer sequences on each side, and the vertical lines mark the start and end of the 24nt repeat sequence. Conservation, icSHAPE and SPEN iCLIP data were from ( Lu et al., 2016 ). One replicate was shown for the eCLIP data of each protein. (F) RBP interaction data for the BCD domain. In the left panel, human 293T cell PARIS data, repeat annotations and eCLIP data are shown for the entire exon 1 ( Lu et al., 2016 ). Vertical lines mark the boundaries of the BCD domain and the internal repetitive sequences in the D-repeat. The right panel shows the average of all D repeats (290nt per unit) and the consensus HNRNP binding site. One replicate was shown for the eCLIP data of each protein. (G) RBP interaction data for the E domain. Parts of exons 1 and 6, and the entire exons 2-5 are shown, together with human 293T PARIS data ( Lu et al., 2016 ). The vertical lines mark the boundaries of the “stem” and “loop” regions of this giant stemloop. One replicate was shown for the eCLIP data of each protein. (H) Schematic RBP interaction model for the E domain. Only the four proteins that mark the “stem” and “loop” are shown. (I) Comparison of protein binding sites on XIST based on eCLIP and the sequence motifs. eCLIP data are normalized against size-matched input in 100nt windows. The density profile was calculated based on sequence motifs in 300nt windows and 50nt steps. See also Figure S2 .

    Techniques Used: Binding Assay, Sequencing, Protein Binding

    XIST RNA structure determines m 6 A modification patterns. (A) m 6 A modfications on the human mature XIST RNA. The RBM15 eCLIP tracks from K562 cells were normalized against input in 100nt windows ( Van Nostrand et al., 2016 ). Then the enrichment on XIST was plotted in two scales: 0-500 and 0-40, to highlight the differences in binding to the 5’ end and the other regions. The HEK293 cell m 6 A iCLIP track was from ( Linder et al., 2015 ). The m 6 A motif (DRACH) density was calculated in 300nt windows and 50nt steps. (B) Gene structure for the alleles. WT and ΔSX (deletion of ∼900bp in the 5’ end of Xist gene) alleles were from ( Wutz et al., 2002 ), under the control of tetracycline inducible promoter. The A-repeat relocation alleles KI5, KI14, KI17 were derived from ΔSX by insertion of the A-repeat in the indicated locations. Two clones were analyzed for KI17. (C) The pipeline for the meRIP-seq analysis. Global analysis of changes in m 6 A modifications was performed on data mapped to the mm10 genome, while targeted analysis of modifications was performed on data converted to the mature mouse Xist transcript (long isoform, with 7 exons). (D) m 6 A modification sites are changed after relocating the A-repeat domain. The mouse Xist RNA PARIS structure model is the same as in Figure 3A . The m 6 A domains are labeled under the genome browser tracks. The Y-axis is the same in each track. All data including the ones with A-repeat insertion at other locations, were mapped to the same wildtype Xist mature RNA. (E-J). Zoom-in view of all the m 6 A domains. Location of the original A repeat is indicated in panel E. The A-repeat knockin locations are indicated in panels F, I and J. Y-axis scales are the same for all tracks in each panel. (K) Quantification of the changes of m 6 A modification relative to wildtype in log scale in the pre-defined m 6 A domains shown in panel D. (L) A model of the role of RNA structures in guiding m 6 A modifications. The A-repeat domain recruits the m 6 A methylase complex to modify sequences that are physically close the domain. The residual modification on Xist after A-repeat deletion was due to its intrinsic ability to recruit m 6 A methylase complex (see the ΔSX tracks in panels D-J). Relocation of the A-repeat to the inside of the large domains (KI5 and KI14) induces local modifications (m6AKI5 and m6AKI14). Relocation of the A-repeat to the end of the transcript (KI17) induces modification in physical proximity (m6AD1, m6AD2, m6AD3 and m6AD4). See also Figure S4 .
    Figure Legend Snippet: XIST RNA structure determines m 6 A modification patterns. (A) m 6 A modfications on the human mature XIST RNA. The RBM15 eCLIP tracks from K562 cells were normalized against input in 100nt windows ( Van Nostrand et al., 2016 ). Then the enrichment on XIST was plotted in two scales: 0-500 and 0-40, to highlight the differences in binding to the 5’ end and the other regions. The HEK293 cell m 6 A iCLIP track was from ( Linder et al., 2015 ). The m 6 A motif (DRACH) density was calculated in 300nt windows and 50nt steps. (B) Gene structure for the alleles. WT and ΔSX (deletion of ∼900bp in the 5’ end of Xist gene) alleles were from ( Wutz et al., 2002 ), under the control of tetracycline inducible promoter. The A-repeat relocation alleles KI5, KI14, KI17 were derived from ΔSX by insertion of the A-repeat in the indicated locations. Two clones were analyzed for KI17. (C) The pipeline for the meRIP-seq analysis. Global analysis of changes in m 6 A modifications was performed on data mapped to the mm10 genome, while targeted analysis of modifications was performed on data converted to the mature mouse Xist transcript (long isoform, with 7 exons). (D) m 6 A modification sites are changed after relocating the A-repeat domain. The mouse Xist RNA PARIS structure model is the same as in Figure 3A . The m 6 A domains are labeled under the genome browser tracks. The Y-axis is the same in each track. All data including the ones with A-repeat insertion at other locations, were mapped to the same wildtype Xist mature RNA. (E-J). Zoom-in view of all the m 6 A domains. Location of the original A repeat is indicated in panel E. The A-repeat knockin locations are indicated in panels F, I and J. Y-axis scales are the same for all tracks in each panel. (K) Quantification of the changes of m 6 A modification relative to wildtype in log scale in the pre-defined m 6 A domains shown in panel D. (L) A model of the role of RNA structures in guiding m 6 A modifications. The A-repeat domain recruits the m 6 A methylase complex to modify sequences that are physically close the domain. The residual modification on Xist after A-repeat deletion was due to its intrinsic ability to recruit m 6 A methylase complex (see the ΔSX tracks in panels D-J). Relocation of the A-repeat to the inside of the large domains (KI5 and KI14) induces local modifications (m6AKI5 and m6AKI14). Relocation of the A-repeat to the end of the transcript (KI17) induces modification in physical proximity (m6AD1, m6AD2, m6AD3 and m6AD4). See also Figure S4 .

    Techniques Used: Modification, Binding Assay, Derivative Assay, Clone Assay, Labeling, Knock-In

    fRIP-seq confirms XIST RNA domains and reveals modular RNA-protein interactions. (A) Schematic diagram of the fRIP-seq experiment. The blue and red lines (R1 and R2, or read1 and read2), 31nt each, represent the paired-end sequenced tags. The gray lines represent the non-sequenced regions of the RNA fragments, each ∼200nt long. (B) Analysis strategy for the fRIP-seq data (see Supplementary Methods for details). Paired-end reads are rearranged and mapped to the genome using STAR, which reveals the non-sequenced fragment as a gap (gray line between the two sequenced tags R1 and R2). The mapped reads were remapped to the mature XIST RNA as a mini-genome to allow gap analysis and visualization. (C) Distribution of gaps or distances between the paired-end sequencing tags (R1 and R2). Most of the tag-pairs are from one RNA fragment, therefore within a short distance of each other (left side). A small fraction of the pairs are far away from each other, therefore most likely to be from two proximally ligated fragments (right side, same distribution, but highlighting the low-frequency long-distance pairs). Five major long pair groups (LGs) can be identified. The long distance distributions were plotted so that the y-axis is 1% that of the short distance distributions. The first biological replicate was shown for each protein and the average read numbers and standard deviations are calculated from all biological replicates (n=2 for EZH2 and n = 3 for the rest). (D) Annotation of the human XIST RNA. XIST exons and repeats, phylogenetic conservation (PhastCons 100 and Placental PhyloP from UCSC), and PARIS data in HEK293 cells are shown ( Lu et al., 2016 ). Long distance groups that correspond to 4 fRIP-seq determined LGs (LG1-LG4) were extracted from PARIS data. (E) Long-distance arcs (tag pairs), coverage of long-distance tag-pairs, and coverage of all tags were shown for four examples (Input1, EZH2, SUZ12 and HNRNPU). These four samples correspond to the ones shown in panel (B). Y-axis scale is indicated in the square brackets. (F) Comparison of the overlapping duplex groups from PARIS and the long-distance groups from EZH2 fRIP-seq. (G) Comparison of the PARIS and EZH2 fRIP-seq long-distance group 2 (LG2), and LG2 extended to the average fRIP-seq fragment size (LG2extend [0-194]). Each side shows a 400nt window. (H) Unsupervised clustering of the XIST-Protein interaction profiles in 100nt windows. A total of 74 samples are clustered, excluding the samples STAG2 (non-specific, as determined by ( Hendrickson et al., 2016 )) and WDR5 (low read numbers). (I) PCA analysis of the profiles in 100nt windows, displaying the top 7 principal components, which explains more than 90% of all variation (top 4 components explain 86%). The percentages on the right represent variation explained by each component. See also Figure S1 .
    Figure Legend Snippet: fRIP-seq confirms XIST RNA domains and reveals modular RNA-protein interactions. (A) Schematic diagram of the fRIP-seq experiment. The blue and red lines (R1 and R2, or read1 and read2), 31nt each, represent the paired-end sequenced tags. The gray lines represent the non-sequenced regions of the RNA fragments, each ∼200nt long. (B) Analysis strategy for the fRIP-seq data (see Supplementary Methods for details). Paired-end reads are rearranged and mapped to the genome using STAR, which reveals the non-sequenced fragment as a gap (gray line between the two sequenced tags R1 and R2). The mapped reads were remapped to the mature XIST RNA as a mini-genome to allow gap analysis and visualization. (C) Distribution of gaps or distances between the paired-end sequencing tags (R1 and R2). Most of the tag-pairs are from one RNA fragment, therefore within a short distance of each other (left side). A small fraction of the pairs are far away from each other, therefore most likely to be from two proximally ligated fragments (right side, same distribution, but highlighting the low-frequency long-distance pairs). Five major long pair groups (LGs) can be identified. The long distance distributions were plotted so that the y-axis is 1% that of the short distance distributions. The first biological replicate was shown for each protein and the average read numbers and standard deviations are calculated from all biological replicates (n=2 for EZH2 and n = 3 for the rest). (D) Annotation of the human XIST RNA. XIST exons and repeats, phylogenetic conservation (PhastCons 100 and Placental PhyloP from UCSC), and PARIS data in HEK293 cells are shown ( Lu et al., 2016 ). Long distance groups that correspond to 4 fRIP-seq determined LGs (LG1-LG4) were extracted from PARIS data. (E) Long-distance arcs (tag pairs), coverage of long-distance tag-pairs, and coverage of all tags were shown for four examples (Input1, EZH2, SUZ12 and HNRNPU). These four samples correspond to the ones shown in panel (B). Y-axis scale is indicated in the square brackets. (F) Comparison of the overlapping duplex groups from PARIS and the long-distance groups from EZH2 fRIP-seq. (G) Comparison of the PARIS and EZH2 fRIP-seq long-distance group 2 (LG2), and LG2 extended to the average fRIP-seq fragment size (LG2extend [0-194]). Each side shows a 400nt window. (H) Unsupervised clustering of the XIST-Protein interaction profiles in 100nt windows. A total of 74 samples are clustered, excluding the samples STAG2 (non-specific, as determined by ( Hendrickson et al., 2016 )) and WDR5 (low read numbers). (I) PCA analysis of the profiles in 100nt windows, displaying the top 7 principal components, which explains more than 90% of all variation (top 4 components explain 86%). The percentages on the right represent variation explained by each component. See also Figure S1 .

    Techniques Used: Sequencing

    Spatial separation of XIST RNP functions in binding chromatin and nuclear lamina. (A) Schematic diagram of the PIRCh method. Orange lines: genomic DNA, light blue lines: RNA. The Y shape represents antibodies against histones. Ctrl: control, IP: Immunoprecipitation. (B) Annotation of the human XIST RNA. XIST exons and repeats, phylogenetic conservation (PhastCons 100 and Placental PhyloP from UCSC), and PARIS data in HEK293 cells are shown, same as Figure 1D . (C) HNRNPU fRIP-seq ( Hendrickson et al. 2016 ) and eCLIP data in human HEK293 cells ( Van Nostrand et al. 2016 ) were normalized against their input controls in 100nt binds. The mouse CIZ1 binding site track was made based on Ridings-Figueroa et al. 2017 , Sunwoo et al. 2017 , showing the enrichment of CIZ1 binding on the E-repeat. The LBR raw data and normalized enrichment ratios (in 100nt bins) were from mouse ES cells ( Chen et al. 2016 ), and then lifted to human XIST coordinates. Human and mouse PIRCh data were all normalized against their own controls, respectively in 100nt bins. The mouse PIRCh data were lifted to human XIST coordinates. (D) Model for the spatial separation of XIST RNP functions in binding the inactive X chromosome and the nuclear lamina, color coded the same way as panel (A).
    Figure Legend Snippet: Spatial separation of XIST RNP functions in binding chromatin and nuclear lamina. (A) Schematic diagram of the PIRCh method. Orange lines: genomic DNA, light blue lines: RNA. The Y shape represents antibodies against histones. Ctrl: control, IP: Immunoprecipitation. (B) Annotation of the human XIST RNA. XIST exons and repeats, phylogenetic conservation (PhastCons 100 and Placental PhyloP from UCSC), and PARIS data in HEK293 cells are shown, same as Figure 1D . (C) HNRNPU fRIP-seq ( Hendrickson et al. 2016 ) and eCLIP data in human HEK293 cells ( Van Nostrand et al. 2016 ) were normalized against their input controls in 100nt binds. The mouse CIZ1 binding site track was made based on Ridings-Figueroa et al. 2017 , Sunwoo et al. 2017 , showing the enrichment of CIZ1 binding on the E-repeat. The LBR raw data and normalized enrichment ratios (in 100nt bins) were from mouse ES cells ( Chen et al. 2016 ), and then lifted to human XIST coordinates. Human and mouse PIRCh data were all normalized against their own controls, respectively in 100nt bins. The mouse PIRCh data were lifted to human XIST coordinates. (D) Model for the spatial separation of XIST RNP functions in binding the inactive X chromosome and the nuclear lamina, color coded the same way as panel (A).

    Techniques Used: Binding Assay, Immunoprecipitation

    5) Product Images from "Polynucleotide phosphorylase promotes the stability and function of Hfq-binding sRNAs by degrading target mRNA-derived fragments"

    Article Title: Polynucleotide phosphorylase promotes the stability and function of Hfq-binding sRNAs by degrading target mRNA-derived fragments

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz616

    PNPase is critical for the decay of specific mRNA-derived fragments. ( A ) A Venn diagram indicating the number of common and uniquely identified differentially regulated genes between the short RNA-seq and mRNA-seq data sets. Numbers represent genes identified by DESeq2 as significantly differentially expressed with a log 2 fold-change in expression of at least 1.5 when comparing the WT (KR10000) and Δ pnp (NRD999) strains. ( B ) RNA-seq read coverage of mRNA and short RNA library preparations of WT (KR10000; black) and Δ pnp (NRD999; red) strains. In the absence of PNPase, numerous short RNA fragments accumulate, including those corresponding to the chiP RBS, dsbB CDS, cfa 5′ UTR, yqaE RBS, hemN 5′ UTR, and miaA CDS. Coverage represents depth per million paired-end fragments and was averaged between two normalized replicates. ( C ) 106 short RNA fragments accumulating in the Δ pnp strain were computationally identified. Gray bars indicate the number of RNA fragments corresponding to each gene feature type (5′ UTR, RBS, CDS, 3′ UTR, IGR). Blue bars represent the number of identified fragments that are associated with RNAs shown to interact with Hfq-binding sRNAs in prior RIL-seq experiments ( 41 ). Orange bars indicate how many RNA fragments would be expected to associate with mRNAs previously shown to interact with Hfq-binding sRNAs in RIL-seq experiments if RNA fragments were chosen at random from the E. coli K12-MG1655 transcriptome. ( D ) Northern blots probed for the presence of the RNA fragments shown in B in the wild-type (KR10000), Δ pnp (NRD999), rph-1 (NRD1138), and rph-1 Δ pnp (NRD1139) strains grown to OD 600 1.0. SsrA served as a loading control. An expanded view of the northern blots is shown in Supplementary Figure S1 .
    Figure Legend Snippet: PNPase is critical for the decay of specific mRNA-derived fragments. ( A ) A Venn diagram indicating the number of common and uniquely identified differentially regulated genes between the short RNA-seq and mRNA-seq data sets. Numbers represent genes identified by DESeq2 as significantly differentially expressed with a log 2 fold-change in expression of at least 1.5 when comparing the WT (KR10000) and Δ pnp (NRD999) strains. ( B ) RNA-seq read coverage of mRNA and short RNA library preparations of WT (KR10000; black) and Δ pnp (NRD999; red) strains. In the absence of PNPase, numerous short RNA fragments accumulate, including those corresponding to the chiP RBS, dsbB CDS, cfa 5′ UTR, yqaE RBS, hemN 5′ UTR, and miaA CDS. Coverage represents depth per million paired-end fragments and was averaged between two normalized replicates. ( C ) 106 short RNA fragments accumulating in the Δ pnp strain were computationally identified. Gray bars indicate the number of RNA fragments corresponding to each gene feature type (5′ UTR, RBS, CDS, 3′ UTR, IGR). Blue bars represent the number of identified fragments that are associated with RNAs shown to interact with Hfq-binding sRNAs in prior RIL-seq experiments ( 41 ). Orange bars indicate how many RNA fragments would be expected to associate with mRNAs previously shown to interact with Hfq-binding sRNAs in RIL-seq experiments if RNA fragments were chosen at random from the E. coli K12-MG1655 transcriptome. ( D ) Northern blots probed for the presence of the RNA fragments shown in B in the wild-type (KR10000), Δ pnp (NRD999), rph-1 (NRD1138), and rph-1 Δ pnp (NRD1139) strains grown to OD 600 1.0. SsrA served as a loading control. An expanded view of the northern blots is shown in Supplementary Figure S1 .

    Techniques Used: Derivative Assay, RNA Sequencing Assay, Expressing, Chromatin Immunoprecipitation, Binding Assay, Northern Blot

    Substitutions in Hfq that block pairing of sRNAs with target mRNAs suppress the defect in sRNA levels that occurs in a Δ pnp strain. Overnight cultures of strain NRD1138 (WT rph-1 ) and derived strains carrying the pnp deletion (Δ pnp , DS070) and harboring mutations resulting in a substitution in a residue in the rim (R17A), distal face (Y25D), or proximal face (Q8A) of Hfq ( hfqR17A , DS060; hfqY25D , NRD1410; hfqQ8A , DS058; Δ pnp hfqR17A , NRD1474; Δ pnp hfqY25D , NRD1478; Δ pnp hfqQ8A , NRD1473) were diluted into fresh LB medium and grown to early exponential phase (OD 600 of 0.3) or late exponential phase (OD 600 of 1.0). For early exponential phase cultures, RyhB and CyaR sRNAs were induced by the addition of 2,2′-dipyridyl or cAMP, respectively. Total RNA was extracted from cultures 15 min after induction and sRNA levels were examined by northern blot probing for RyhB ( A ), GcvB ( C ), MgrR ( E ), or CyaR ( G ) as described in Materials and Methods. For cultures grown to late exponential phase, total RNA was immediately extracted as described above from cultures for each strain after it reached an OD 600 of 1.0. RyhB ( B ), GcvB ( D ), MgrR ( F ) and CyaR ( H ) were measured by northern blot analysis. sRNA levels were normalized to the control RNA SsrA, the level of each sRNA in WT was set to 100%, and sRNA levels were scaled relative to WT. The results presented in the bar graph represent the mean of three independent experiments and the error bars represent the standard error of the mean.
    Figure Legend Snippet: Substitutions in Hfq that block pairing of sRNAs with target mRNAs suppress the defect in sRNA levels that occurs in a Δ pnp strain. Overnight cultures of strain NRD1138 (WT rph-1 ) and derived strains carrying the pnp deletion (Δ pnp , DS070) and harboring mutations resulting in a substitution in a residue in the rim (R17A), distal face (Y25D), or proximal face (Q8A) of Hfq ( hfqR17A , DS060; hfqY25D , NRD1410; hfqQ8A , DS058; Δ pnp hfqR17A , NRD1474; Δ pnp hfqY25D , NRD1478; Δ pnp hfqQ8A , NRD1473) were diluted into fresh LB medium and grown to early exponential phase (OD 600 of 0.3) or late exponential phase (OD 600 of 1.0). For early exponential phase cultures, RyhB and CyaR sRNAs were induced by the addition of 2,2′-dipyridyl or cAMP, respectively. Total RNA was extracted from cultures 15 min after induction and sRNA levels were examined by northern blot probing for RyhB ( A ), GcvB ( C ), MgrR ( E ), or CyaR ( G ) as described in Materials and Methods. For cultures grown to late exponential phase, total RNA was immediately extracted as described above from cultures for each strain after it reached an OD 600 of 1.0. RyhB ( B ), GcvB ( D ), MgrR ( F ) and CyaR ( H ) were measured by northern blot analysis. sRNA levels were normalized to the control RNA SsrA, the level of each sRNA in WT was set to 100%, and sRNA levels were scaled relative to WT. The results presented in the bar graph represent the mean of three independent experiments and the error bars represent the standard error of the mean.

    Techniques Used: Blocking Assay, Derivative Assay, Northern Blot

    Substitutions in Hfq residues critical for binding mRNA targets suppress the stability defect of sRNAs in a Δ pnp strain. Overnight cultures of strain NRD1138 (WT rph-1 ) and derived strains carrying the pnp deletion (Δ pnp , DS070) and harboring mutations resulting in a substitution in a residue in the rim (R17A) or distal face (Y25D) of Hfq ( hfqR17A , DS060; hfqY25D , NRD1410; Δ pnp hfqR17A , NRD1474; Δ pnp hfqY25D , NRD1478) were diluted into fresh LB medium and grown to early exponential phase (OD 600 of 0.3). RyhB and CyaR sRNAs were induced for 15 min by the addition of 2,2′-dipyridyl or cAMP, respectively. Subsequently, rifampicin RNA stability time courses were performed as described for Figure 3 . sRNA levels were examined by northern blot probing for RyhB ( A, B ), GcvB ( C, D ), MgrR ( E, F ), or CyaR ( G, H ), with representative blots shown. GcvB and MgrR levels were measured by northern blot analysis using RNA samples from the RyhB induction experiment described above. CyaR levels for the Δ pnp hfqY25D strain were too low at the 4 and 6 min time points to accurately measure. sRNA levels were normalized to the control RNA SsrA. The results presented in the graphs represent the mean of three independent experiments, and the error bars represent the standard error of the mean.
    Figure Legend Snippet: Substitutions in Hfq residues critical for binding mRNA targets suppress the stability defect of sRNAs in a Δ pnp strain. Overnight cultures of strain NRD1138 (WT rph-1 ) and derived strains carrying the pnp deletion (Δ pnp , DS070) and harboring mutations resulting in a substitution in a residue in the rim (R17A) or distal face (Y25D) of Hfq ( hfqR17A , DS060; hfqY25D , NRD1410; Δ pnp hfqR17A , NRD1474; Δ pnp hfqY25D , NRD1478) were diluted into fresh LB medium and grown to early exponential phase (OD 600 of 0.3). RyhB and CyaR sRNAs were induced for 15 min by the addition of 2,2′-dipyridyl or cAMP, respectively. Subsequently, rifampicin RNA stability time courses were performed as described for Figure 3 . sRNA levels were examined by northern blot probing for RyhB ( A, B ), GcvB ( C, D ), MgrR ( E, F ), or CyaR ( G, H ), with representative blots shown. GcvB and MgrR levels were measured by northern blot analysis using RNA samples from the RyhB induction experiment described above. CyaR levels for the Δ pnp hfqY25D strain were too low at the 4 and 6 min time points to accurately measure. sRNA levels were normalized to the control RNA SsrA. The results presented in the graphs represent the mean of three independent experiments, and the error bars represent the standard error of the mean.

    Techniques Used: Binding Assay, Derivative Assay, Northern Blot

    mRNA-derived fragments that accumulate in the absence of PNPase interact with Hfq. ( A ) A Venn diagram indicating the number of common and uniquely identified differentially regulated genes between the Hfq input and co-IP data sets. Numbers represent genes identified by DESeq2 as significantly differentially expressed with a log 2 fold-change in expression of at least 1.5 when comparing the WT (KR10000) and Δ pnp (NRD999) strains. ( B ) RNA-seq read coverage of short RNA library preparations of input RNA and RNA co-immunoprecipitated with Hfq for the WT (KR10000; black) and Δ pnp (NRD999; red) strains for the same regions shown in Figure 1B . Coverage represents depth per million paired-end fragments and was averaged between two normalized replicates. ( C ) Input and Hfq co-immunoprecipitation fractions probed by northern blot for the presence of the RNA fragments shown in B in the wild-type (KR10000) and Δ pnp (NRD999) strains. SsrA served as a loading control. An expanded view of the northern blots is shown in Supplementary Figure S2 .
    Figure Legend Snippet: mRNA-derived fragments that accumulate in the absence of PNPase interact with Hfq. ( A ) A Venn diagram indicating the number of common and uniquely identified differentially regulated genes between the Hfq input and co-IP data sets. Numbers represent genes identified by DESeq2 as significantly differentially expressed with a log 2 fold-change in expression of at least 1.5 when comparing the WT (KR10000) and Δ pnp (NRD999) strains. ( B ) RNA-seq read coverage of short RNA library preparations of input RNA and RNA co-immunoprecipitated with Hfq for the WT (KR10000; black) and Δ pnp (NRD999; red) strains for the same regions shown in Figure 1B . Coverage represents depth per million paired-end fragments and was averaged between two normalized replicates. ( C ) Input and Hfq co-immunoprecipitation fractions probed by northern blot for the presence of the RNA fragments shown in B in the wild-type (KR10000) and Δ pnp (NRD999) strains. SsrA served as a loading control. An expanded view of the northern blots is shown in Supplementary Figure S2 .

    Techniques Used: Derivative Assay, Co-Immunoprecipitation Assay, Expressing, RNA Sequencing Assay, Immunoprecipitation, Northern Blot

    6) Product Images from "Small-molecule targeting of oncogenic FTO demethylase in acute myeloid leukemia"

    Article Title: Small-molecule targeting of oncogenic FTO demethylase in acute myeloid leukemia

    Journal: Cancer cell

    doi: 10.1016/j.ccell.2019.03.006

    Transcriptome-wide RNA-seq assays to identify potential targets of FTO inhibitors in AML cells.
    Figure Legend Snippet: Transcriptome-wide RNA-seq assays to identify potential targets of FTO inhibitors in AML cells.

    Techniques Used: RNA Sequencing Assay

    7) Product Images from "m6A modification suppresses ocular melanoma through modulating HINT2 mRNA translation"

    Article Title: m6A modification suppresses ocular melanoma through modulating HINT2 mRNA translation

    Journal: Molecular Cancer

    doi: 10.1186/s12943-019-1088-x

    Dynamic m 6 A modifications were highly associated with ocular melanoma tumorigenesis. a Top enriched motifs within m 6 A peaks identified in ocular melanoma and normal cells. b Distribution of m 6 A sites along the length of mRNA transcripts. c Pie charts showing the m 6 A peak distribution in different RNA regions (CDS, 5′ UTR, 3′ UTR and stop codon) in ocular melanoma and normal cells. d The percentage of methylated genes with 1, 2, 3, 4 or more than 5 peaks per gene in each cell line. e GO enrichment map of m 6 A-containing genes in ocular melanoma and normal cells. f KEGG pathway analysis of m 6 A-modified genes in ocular melanoma and normal cells
    Figure Legend Snippet: Dynamic m 6 A modifications were highly associated with ocular melanoma tumorigenesis. a Top enriched motifs within m 6 A peaks identified in ocular melanoma and normal cells. b Distribution of m 6 A sites along the length of mRNA transcripts. c Pie charts showing the m 6 A peak distribution in different RNA regions (CDS, 5′ UTR, 3′ UTR and stop codon) in ocular melanoma and normal cells. d The percentage of methylated genes with 1, 2, 3, 4 or more than 5 peaks per gene in each cell line. e GO enrichment map of m 6 A-containing genes in ocular melanoma and normal cells. f KEGG pathway analysis of m 6 A-modified genes in ocular melanoma and normal cells

    Techniques Used: Methylation, Modification

    8) Product Images from "Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma"

    Article Title: Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma

    Journal: Nature

    doi: 10.1038/nature24014

    NLGN3 shedding from glioma cells is regulated by NLGN3 exposure and is mediated by ADAM10 a , NLGN3 Western blot illustrating neuroligin-3 secreted into CM from optogenetically stimulated Thy1::ChR2 ; NSG cortical slices (ChR2 stim slice) or SU-DIPGXIII xenograft-bearing Thy1::ChR2 ; NSG cortical slices (ChR2 stim slice with xenograft). Performed in biological duplicate. b , NLGN3 western blot illustrating neuroligin-3 secreted into CM from wild type brain slices (WT), WT brain slices bearing xenografts of adult GBM SU-GBM035 (WT + xeno), or from Nlgn3 knockout brains bearing SU-GBM035 xenografts (Nlgn3 KO + xeno) in the absence (left 3 lanes) or presence (right 3 lanes) of 200 nM ADAM10 inhibitor GI254023X (+ADAM10i). Performed in biological triplicate. c , NLGN3 Western illustrating glioma cell secretion of NLGN3 in vitro at baseline medium conditions (aCSF), following exposure to recombinant NLGN3 with subsequent washing (NLGN3), at baseline medium conditions in the presence of ADAM10 inhibitor GI254023X (aCSF + ADAM10i) or following NLGN3 exposure in the presence of ADAM10 inhibitor (NLGN3+ADAM10i). Performed in biological triplicate. d , mRNA expression levels of ADAM10 in primary tumor and cultures of DIPG by RNA-seq with values reported as FPKM 12 , 28 (left; n =8 primary samples, n =7 culture samples) and in 493 individual adult glioblastoma samples from TCGA 29 (right). Values are reported as robust multi-array averages (RMA; right). Boxes show the median, 25th and 75th percentiles, error bars show the minima and maxima.
    Figure Legend Snippet: NLGN3 shedding from glioma cells is regulated by NLGN3 exposure and is mediated by ADAM10 a , NLGN3 Western blot illustrating neuroligin-3 secreted into CM from optogenetically stimulated Thy1::ChR2 ; NSG cortical slices (ChR2 stim slice) or SU-DIPGXIII xenograft-bearing Thy1::ChR2 ; NSG cortical slices (ChR2 stim slice with xenograft). Performed in biological duplicate. b , NLGN3 western blot illustrating neuroligin-3 secreted into CM from wild type brain slices (WT), WT brain slices bearing xenografts of adult GBM SU-GBM035 (WT + xeno), or from Nlgn3 knockout brains bearing SU-GBM035 xenografts (Nlgn3 KO + xeno) in the absence (left 3 lanes) or presence (right 3 lanes) of 200 nM ADAM10 inhibitor GI254023X (+ADAM10i). Performed in biological triplicate. c , NLGN3 Western illustrating glioma cell secretion of NLGN3 in vitro at baseline medium conditions (aCSF), following exposure to recombinant NLGN3 with subsequent washing (NLGN3), at baseline medium conditions in the presence of ADAM10 inhibitor GI254023X (aCSF + ADAM10i) or following NLGN3 exposure in the presence of ADAM10 inhibitor (NLGN3+ADAM10i). Performed in biological triplicate. d , mRNA expression levels of ADAM10 in primary tumor and cultures of DIPG by RNA-seq with values reported as FPKM 12 , 28 (left; n =8 primary samples, n =7 culture samples) and in 493 individual adult glioblastoma samples from TCGA 29 (right). Values are reported as robust multi-array averages (RMA; right). Boxes show the median, 25th and 75th percentiles, error bars show the minima and maxima.

    Techniques Used: Western Blot, Knock-Out, In Vitro, Recombinant, Expressing, RNA Sequencing Assay

    9) Product Images from "A central role for PI3K-AKT signaling pathway in linking SAMHD1-deficiency to the type I interferon signature"

    Article Title: A central role for PI3K-AKT signaling pathway in linking SAMHD1-deficiency to the type I interferon signature

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-18308-8

    Accumulated RNAs in SAMHD1 -deficient cells function as immune stimuli. ( A , B ) PMA-differentiated wild-type THP-1 cells were stimulated with poly dA:dT, poly I:C, an equal amount (5 μg/ml) of isolated total DNA and RNA from wild-type and SAMHD1 -deficient cells, or left unstimulated ( A ). Total RNA isolated from wild-type and SAMHD1 -deficient cells were further size-fractionated and an equal amount of RNA from each fraction was used to stimulate PMA-differentiated wild-type THP-1 cells ( B ), followed by qRT-PCR analysis of IFN-α , IFN-β , IFITM1 and IL6 mRNA levels. ( C , D ) In vitro RNase activity assay for SAMHD1 immunopurified from undifferentiated THP-1 cells using A20 single-stranded RNA substrates. An isotype-matched control anti-IgG and anti-SAMHD1 antibodies were used for immunopurification. THP1 cells were infected with serial dilution of Vpx-loaded or control SIV VLPs ( D ). ( E ) qRT-PCR analysis of IFN-α in wild-type and SAMHD1 -deficient cells reconstituted with indicated SAMHD1 wild-type and mutant constructs. ( F ) Autoradiography of SAMHD1-RNA complex and western blotting of SAMHD1 protein immunoprecipitated from SAMHD1 CLIP. ( G ) Pie chart showing the distribution of statistically significant peaks (q
    Figure Legend Snippet: Accumulated RNAs in SAMHD1 -deficient cells function as immune stimuli. ( A , B ) PMA-differentiated wild-type THP-1 cells were stimulated with poly dA:dT, poly I:C, an equal amount (5 μg/ml) of isolated total DNA and RNA from wild-type and SAMHD1 -deficient cells, or left unstimulated ( A ). Total RNA isolated from wild-type and SAMHD1 -deficient cells were further size-fractionated and an equal amount of RNA from each fraction was used to stimulate PMA-differentiated wild-type THP-1 cells ( B ), followed by qRT-PCR analysis of IFN-α , IFN-β , IFITM1 and IL6 mRNA levels. ( C , D ) In vitro RNase activity assay for SAMHD1 immunopurified from undifferentiated THP-1 cells using A20 single-stranded RNA substrates. An isotype-matched control anti-IgG and anti-SAMHD1 antibodies were used for immunopurification. THP1 cells were infected with serial dilution of Vpx-loaded or control SIV VLPs ( D ). ( E ) qRT-PCR analysis of IFN-α in wild-type and SAMHD1 -deficient cells reconstituted with indicated SAMHD1 wild-type and mutant constructs. ( F ) Autoradiography of SAMHD1-RNA complex and western blotting of SAMHD1 protein immunoprecipitated from SAMHD1 CLIP. ( G ) Pie chart showing the distribution of statistically significant peaks (q

    Techniques Used: Isolation, Quantitative RT-PCR, In Vitro, Activity Assay, Immu-Puri, Infection, Serial Dilution, Mutagenesis, Construct, Autoradiography, Western Blot, Immunoprecipitation, Cross-linking Immunoprecipitation

    10) Product Images from "A central role for PI3K-AKT signaling pathway in linking SAMHD1-deficiency to the type I interferon signature"

    Article Title: A central role for PI3K-AKT signaling pathway in linking SAMHD1-deficiency to the type I interferon signature

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-18308-8

    Accumulated RNAs in SAMHD1 -deficient cells function as immune stimuli. ( A , B ) PMA-differentiated wild-type THP-1 cells were stimulated with poly dA:dT, poly I:C, an equal amount (5 μg/ml) of isolated total DNA and RNA from wild-type and SAMHD1 -deficient cells, or left unstimulated ( A ). Total RNA isolated from wild-type and SAMHD1 -deficient cells were further size-fractionated and an equal amount of RNA from each fraction was used to stimulate PMA-differentiated wild-type THP-1 cells ( B ), followed by qRT-PCR analysis of IFN-α , IFN-β , IFITM1 and IL6 mRNA levels. ( C , D ) In vitro RNase activity assay for SAMHD1 immunopurified from undifferentiated THP-1 cells using A20 single-stranded RNA substrates. An isotype-matched control anti-IgG and anti-SAMHD1 antibodies were used for immunopurification. THP1 cells were infected with serial dilution of Vpx-loaded or control SIV VLPs ( D ). ( E ) qRT-PCR analysis of IFN-α in wild-type and SAMHD1 -deficient cells reconstituted with indicated SAMHD1 wild-type and mutant constructs. ( F ) Autoradiography of SAMHD1-RNA complex and western blotting of SAMHD1 protein immunoprecipitated from SAMHD1 CLIP. ( G ) Pie chart showing the distribution of statistically significant peaks (q
    Figure Legend Snippet: Accumulated RNAs in SAMHD1 -deficient cells function as immune stimuli. ( A , B ) PMA-differentiated wild-type THP-1 cells were stimulated with poly dA:dT, poly I:C, an equal amount (5 μg/ml) of isolated total DNA and RNA from wild-type and SAMHD1 -deficient cells, or left unstimulated ( A ). Total RNA isolated from wild-type and SAMHD1 -deficient cells were further size-fractionated and an equal amount of RNA from each fraction was used to stimulate PMA-differentiated wild-type THP-1 cells ( B ), followed by qRT-PCR analysis of IFN-α , IFN-β , IFITM1 and IL6 mRNA levels. ( C , D ) In vitro RNase activity assay for SAMHD1 immunopurified from undifferentiated THP-1 cells using A20 single-stranded RNA substrates. An isotype-matched control anti-IgG and anti-SAMHD1 antibodies were used for immunopurification. THP1 cells were infected with serial dilution of Vpx-loaded or control SIV VLPs ( D ). ( E ) qRT-PCR analysis of IFN-α in wild-type and SAMHD1 -deficient cells reconstituted with indicated SAMHD1 wild-type and mutant constructs. ( F ) Autoradiography of SAMHD1-RNA complex and western blotting of SAMHD1 protein immunoprecipitated from SAMHD1 CLIP. ( G ) Pie chart showing the distribution of statistically significant peaks (q

    Techniques Used: Isolation, Quantitative RT-PCR, In Vitro, Activity Assay, Immu-Puri, Infection, Serial Dilution, Mutagenesis, Construct, Autoradiography, Western Blot, Immunoprecipitation, Cross-linking Immunoprecipitation

    11) Product Images from "Small-molecule targeting of oncogenic FTO demethylase in acute myeloid leukemia"

    Article Title: Small-molecule targeting of oncogenic FTO demethylase in acute myeloid leukemia

    Journal: Cancer cell

    doi: 10.1016/j.ccell.2019.03.006

    Transcriptome-wide RNA-seq assays to identify potential targets of FTO inhibitors in AML cells.
    Figure Legend Snippet: Transcriptome-wide RNA-seq assays to identify potential targets of FTO inhibitors in AML cells.

    Techniques Used: RNA Sequencing Assay

    Related Articles

    Irradiation:

    Article Title: m6A modification suppresses ocular melanoma through modulating HINT2 mRNA translation
    Article Snippet: .. Briefly, 100 ng of mRNA was isolated from the tumor (OCM1, OCM1a, OM431, CRMM1 and CM2005.1) and normal (PIG1) cell lines using the Dynabeads mRNA Purification Kit (Life Technologies, 61,006), fragmented to ~ 100 nucleotides using fragmentation reagent (Life Technologies, AM8740), and incubated with 5 μg of an anti-m6 A antibody (Abcam, ab151230) in 450 μL of IP buffer (50 mM Tris, 100 mM NaCl, 0.05% NP-40, adjusted to pH 7.4) under gentle rotation at 4 °C for 2 h. The mixture was transferred to a clear flat-bottom 96-well plate (Corning) on ice and irradiated three times with 0.15 J/cm2 at 254 nm using a CL-1000 Ultraviolet Crosslinker (UVP). .. After irradiation, the mixture was collected and incubated with 50 μL of prewashed Dynabeads Protein A (Life Technologies, 1001D) at 4 °C for 2 h. After extensive washing twice with high-salt buffer (50 mM Tris, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, adjusted to pH 7.4) and twice with IP buffer, the samples on the beads were subjected to dephosphorylation with T4 PNK (NEB, M0201 L) at 37 °C for 20 min.

    Methylation:

    Article Title: m6A methylation controls pluripotency of porcine induced pluripotent stem cells by targeting SOCS3/JAK2/STAT3 pathway in a YTHDF1/YTHDF2-orchestrated manner
    Article Snippet: .. Methylated RNA Immunoprecipitation coupled with quantitative real-time PCR (MeRIP-qPCR) mRNA was prepared as described above, and fragmented using Ambion RNA Fragmentation reagent (Ambion, Carlsbad, CA, USA) at 70°C for 15 min. A small portion (10%) of the RNA fragments was collected to be used as input sample. ..

    Isolation:

    Article Title: Small-molecule targeting of oncogenic FTO demethylase in acute myeloid leukemia
    Article Snippet: .. Poly(A)+ RNA was enriched from total RNA samples isolated from DMSO and FB23-2 treated MONOMAC6 AML cells for 72 hr and randomly fragmented with RNA fragmentation reagents (Ambion). .. Poly(A)+ RNA was enriched from total RNA samples isolated from DMSO and FB23-2 treated MONOMAC6 AML cells for 72 hr and randomly fragmented with RNA fragmentation reagents (Ambion).

    Article Title: m6A modification suppresses ocular melanoma through modulating HINT2 mRNA translation
    Article Snippet: .. Briefly, 100 ng of mRNA was isolated from the tumor (OCM1, OCM1a, OM431, CRMM1 and CM2005.1) and normal (PIG1) cell lines using the Dynabeads mRNA Purification Kit (Life Technologies, 61,006), fragmented to ~ 100 nucleotides using fragmentation reagent (Life Technologies, AM8740), and incubated with 5 μg of an anti-m6 A antibody (Abcam, ab151230) in 450 μL of IP buffer (50 mM Tris, 100 mM NaCl, 0.05% NP-40, adjusted to pH 7.4) under gentle rotation at 4 °C for 2 h. The mixture was transferred to a clear flat-bottom 96-well plate (Corning) on ice and irradiated three times with 0.15 J/cm2 at 254 nm using a CL-1000 Ultraviolet Crosslinker (UVP). .. After irradiation, the mixture was collected and incubated with 50 μL of prewashed Dynabeads Protein A (Life Technologies, 1001D) at 4 °C for 2 h. After extensive washing twice with high-salt buffer (50 mM Tris, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, adjusted to pH 7.4) and twice with IP buffer, the samples on the beads were subjected to dephosphorylation with T4 PNK (NEB, M0201 L) at 37 °C for 20 min.

    Purification:

    Article Title: A central role for PI3K-AKT signaling pathway in linking SAMHD1-deficiency to the type I interferon signature
    Article Snippet: .. After RNA fragmentation by RNA fragmentation reagents (Ambion), RNAs of 30–60 nucleotides were purified and ligated with 3′ and 5′ adaptors using T4 RNA Ligase 2, truncated K227Q, and T4 RNA ligase, respectively. .. 5′ and 3′ adaptor ligated RNAs were reverse transcribed by using the RNA RT primer (RTP; 5′-GCCTTGGCACCCGAGAATTCCA-3′, Integrated DNA Technologies).

    Article Title: m6A modification suppresses ocular melanoma through modulating HINT2 mRNA translation
    Article Snippet: .. Briefly, 100 ng of mRNA was isolated from the tumor (OCM1, OCM1a, OM431, CRMM1 and CM2005.1) and normal (PIG1) cell lines using the Dynabeads mRNA Purification Kit (Life Technologies, 61,006), fragmented to ~ 100 nucleotides using fragmentation reagent (Life Technologies, AM8740), and incubated with 5 μg of an anti-m6 A antibody (Abcam, ab151230) in 450 μL of IP buffer (50 mM Tris, 100 mM NaCl, 0.05% NP-40, adjusted to pH 7.4) under gentle rotation at 4 °C for 2 h. The mixture was transferred to a clear flat-bottom 96-well plate (Corning) on ice and irradiated three times with 0.15 J/cm2 at 254 nm using a CL-1000 Ultraviolet Crosslinker (UVP). .. After irradiation, the mixture was collected and incubated with 50 μL of prewashed Dynabeads Protein A (Life Technologies, 1001D) at 4 °C for 2 h. After extensive washing twice with high-salt buffer (50 mM Tris, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, adjusted to pH 7.4) and twice with IP buffer, the samples on the beads were subjected to dephosphorylation with T4 PNK (NEB, M0201 L) at 37 °C for 20 min.

    Real-time Polymerase Chain Reaction:

    Article Title: m6A methylation controls pluripotency of porcine induced pluripotent stem cells by targeting SOCS3/JAK2/STAT3 pathway in a YTHDF1/YTHDF2-orchestrated manner
    Article Snippet: .. Methylated RNA Immunoprecipitation coupled with quantitative real-time PCR (MeRIP-qPCR) mRNA was prepared as described above, and fragmented using Ambion RNA Fragmentation reagent (Ambion, Carlsbad, CA, USA) at 70°C for 15 min. A small portion (10%) of the RNA fragments was collected to be used as input sample. ..

    Immunoprecipitation:

    Article Title: m6A methylation controls pluripotency of porcine induced pluripotent stem cells by targeting SOCS3/JAK2/STAT3 pathway in a YTHDF1/YTHDF2-orchestrated manner
    Article Snippet: .. Methylated RNA Immunoprecipitation coupled with quantitative real-time PCR (MeRIP-qPCR) mRNA was prepared as described above, and fragmented using Ambion RNA Fragmentation reagent (Ambion, Carlsbad, CA, USA) at 70°C for 15 min. A small portion (10%) of the RNA fragments was collected to be used as input sample. ..

    Article Title: Selective termination of lnc RNA transcription promotes heterochromatin silencing and cell differentiation
    Article Snippet: .. Immunoprecipitated RNA was fragmented 200–300nt long using RNA Fragmentation Reagent Kit from (Ambion). .. Fragmented RNA was 5′ phosphorylated using T4 Polynucleotide Kinase (Fermentas) and ligated to the 5′ adaptor using the T4 RNA ligase (Fermentas).

    Article Title: Transcriptome-Wide Mapping of 5-methylcytidine RNA Modifications in Bacteria, Archaea, and Yeast Reveals m5C within Archaeal mRNAs
    Article Snippet: .. RNA immunoprecipitation Sulfolobus solfataricus total RNA was chemically fragmented (Ambion, AM8740) to an average size of ∼100 bp and was subjected to immunoprecipitation with an anti m5 C monoclonal antibody (Diagenode, MAb-081-010) or anti-hm5 C polyclonal antibody (Diagenode, pAb-HMC-050) based on the protocol described in ref , using two rounds of IP. .. The precipitated RNA was used for dsDNA Illumina library construction.

    Incubation:

    Article Title: m6A modification suppresses ocular melanoma through modulating HINT2 mRNA translation
    Article Snippet: .. Briefly, 100 ng of mRNA was isolated from the tumor (OCM1, OCM1a, OM431, CRMM1 and CM2005.1) and normal (PIG1) cell lines using the Dynabeads mRNA Purification Kit (Life Technologies, 61,006), fragmented to ~ 100 nucleotides using fragmentation reagent (Life Technologies, AM8740), and incubated with 5 μg of an anti-m6 A antibody (Abcam, ab151230) in 450 μL of IP buffer (50 mM Tris, 100 mM NaCl, 0.05% NP-40, adjusted to pH 7.4) under gentle rotation at 4 °C for 2 h. The mixture was transferred to a clear flat-bottom 96-well plate (Corning) on ice and irradiated three times with 0.15 J/cm2 at 254 nm using a CL-1000 Ultraviolet Crosslinker (UVP). .. After irradiation, the mixture was collected and incubated with 50 μL of prewashed Dynabeads Protein A (Life Technologies, 1001D) at 4 °C for 2 h. After extensive washing twice with high-salt buffer (50 mM Tris, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, adjusted to pH 7.4) and twice with IP buffer, the samples on the beads were subjected to dephosphorylation with T4 PNK (NEB, M0201 L) at 37 °C for 20 min.

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    Thermo Fisher rna fragmentation reagents
    Targeted KO-first targeting strategy in Rhbdf1 (A3) generates novel transcripts and N-terminally truncated functional proteins a. Schematic of the strategy used by Li et al. for generation of Rhbdf1 −/− homozygous mutant mice; the Rhbdf1 KO-first allele was crossed to Flp recombinase mice to remove the FRT-flanked “lacZ reporter and a neomycin resistance (neo) gene” to generate conditional-ready mice, which were later crossed with cre transgenic mice to excise the floxed gene segment (exons 4-11), generating Rhbdf1 −/− homozygous mutant mice (hereafter referred as viable2 mice, Rhbdf1 v2/v2 mice). b. Whole-exome sequencing of spleen tissue from Rhbdf1 v2/v2 mice showing loss of exons 4 through 11 in Rhbdf1 v2/v2 mutant mice. c. RT-PCR on spleens from Rhbdf1 +/+ and Rhbdf1 v2/v2 mutant mice using primers to amplify exons 6 through 8, exons 7 through 10, and exons 16 and 17. Exons 4-11 are deleted in Rhbdf1 v2/v2 mutant mice; hence no amplicons were generated using either exon 6 forward and exon 8 reverse, or exon 7 forward and exon 10 reverse, primers. However, exon 16 forward and exon 17 reverse primers generated a 211-bp product. d. <t>RNA-Seq</t> analysis of spleens from Rhbdf1 v2/v2 mutant mice indicating loss of exons 4 through 11; however, there is strong evidence for mutant <t>mRNA,</t> as indicated by the presence of the rest of the transcript, which encodes exons 12 through 18 and is not degraded by the nonsense-mediated decay mechanism. e. Schematic representation of exons and introns in the Rhbdf1 v2/v2 mutant allele. 5’ RACE using a gene-specific exon 16-17 fusion primer (GSP) was used to obtain 5’ ends of the Rhbdf1 v2/v2 mutant mRNA. We identified several novel mutant mRNAs with different translation initiation sites that could potentially generate N-terminally truncated RHBDF1 mutant proteins. See supplemental figures for variant protein and 5’ UTR sequences. Alternative exons are indicated as red boxes; predicted translation initiation sites are indicated by “START,” and termination codons are indicated by “STOP.” f. C-terminal Myc-DDK-tagged Rhbdf1 v2/v2 variant protein 1 (lanes 1, 2) or variant protein 2 (lanes 3,4), or empty vector (lanes 5, 6) were transiently expressed in 293T cells, and cell lysates were analyzed using western blotting with FLAG-specific antibody. After visualization of blots with a G:Box chemiluminescent imaging system, blots were washed, blocked in 5% nonfat dry milk, and re-probed with anti-actin antibody. g. Rescue of phenotype in Rhbdf1 −/− MEFs. Rhbdf1 +/+ (top) and Rhbdf1 −/− (bottom) MEFs were transiently transfected with 2 μg of either variant 1 or variant 2 vectors, or an empty vector, using Lipofectamine LTX. 48 h post-transfection, cells were stimulated overnight with either DMSO or 100 nM PMA, and cell-culture supernatants were analyzed using a mouse AREG ELISA kit. Data represent mean ± S.D; *p
    Rna Fragmentation Reagents, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 203 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Targeted KO-first targeting strategy in Rhbdf1 (A3) generates novel transcripts and N-terminally truncated functional proteins a. Schematic of the strategy used by Li et al. for generation of Rhbdf1 −/− homozygous mutant mice; the Rhbdf1 KO-first allele was crossed to Flp recombinase mice to remove the FRT-flanked “lacZ reporter and a neomycin resistance (neo) gene” to generate conditional-ready mice, which were later crossed with cre transgenic mice to excise the floxed gene segment (exons 4-11), generating Rhbdf1 −/− homozygous mutant mice (hereafter referred as viable2 mice, Rhbdf1 v2/v2 mice). b. Whole-exome sequencing of spleen tissue from Rhbdf1 v2/v2 mice showing loss of exons 4 through 11 in Rhbdf1 v2/v2 mutant mice. c. RT-PCR on spleens from Rhbdf1 +/+ and Rhbdf1 v2/v2 mutant mice using primers to amplify exons 6 through 8, exons 7 through 10, and exons 16 and 17. Exons 4-11 are deleted in Rhbdf1 v2/v2 mutant mice; hence no amplicons were generated using either exon 6 forward and exon 8 reverse, or exon 7 forward and exon 10 reverse, primers. However, exon 16 forward and exon 17 reverse primers generated a 211-bp product. d. RNA-Seq analysis of spleens from Rhbdf1 v2/v2 mutant mice indicating loss of exons 4 through 11; however, there is strong evidence for mutant mRNA, as indicated by the presence of the rest of the transcript, which encodes exons 12 through 18 and is not degraded by the nonsense-mediated decay mechanism. e. Schematic representation of exons and introns in the Rhbdf1 v2/v2 mutant allele. 5’ RACE using a gene-specific exon 16-17 fusion primer (GSP) was used to obtain 5’ ends of the Rhbdf1 v2/v2 mutant mRNA. We identified several novel mutant mRNAs with different translation initiation sites that could potentially generate N-terminally truncated RHBDF1 mutant proteins. See supplemental figures for variant protein and 5’ UTR sequences. Alternative exons are indicated as red boxes; predicted translation initiation sites are indicated by “START,” and termination codons are indicated by “STOP.” f. C-terminal Myc-DDK-tagged Rhbdf1 v2/v2 variant protein 1 (lanes 1, 2) or variant protein 2 (lanes 3,4), or empty vector (lanes 5, 6) were transiently expressed in 293T cells, and cell lysates were analyzed using western blotting with FLAG-specific antibody. After visualization of blots with a G:Box chemiluminescent imaging system, blots were washed, blocked in 5% nonfat dry milk, and re-probed with anti-actin antibody. g. Rescue of phenotype in Rhbdf1 −/− MEFs. Rhbdf1 +/+ (top) and Rhbdf1 −/− (bottom) MEFs were transiently transfected with 2 μg of either variant 1 or variant 2 vectors, or an empty vector, using Lipofectamine LTX. 48 h post-transfection, cells were stimulated overnight with either DMSO or 100 nM PMA, and cell-culture supernatants were analyzed using a mouse AREG ELISA kit. Data represent mean ± S.D; *p

    Journal: bioRxiv

    Article Title: Genes adapt to outsmart gene targeting strategies in mutant mouse strains by skipping exons to reinitiate transcription and translation

    doi: 10.1101/2020.04.22.041087

    Figure Lengend Snippet: Targeted KO-first targeting strategy in Rhbdf1 (A3) generates novel transcripts and N-terminally truncated functional proteins a. Schematic of the strategy used by Li et al. for generation of Rhbdf1 −/− homozygous mutant mice; the Rhbdf1 KO-first allele was crossed to Flp recombinase mice to remove the FRT-flanked “lacZ reporter and a neomycin resistance (neo) gene” to generate conditional-ready mice, which were later crossed with cre transgenic mice to excise the floxed gene segment (exons 4-11), generating Rhbdf1 −/− homozygous mutant mice (hereafter referred as viable2 mice, Rhbdf1 v2/v2 mice). b. Whole-exome sequencing of spleen tissue from Rhbdf1 v2/v2 mice showing loss of exons 4 through 11 in Rhbdf1 v2/v2 mutant mice. c. RT-PCR on spleens from Rhbdf1 +/+ and Rhbdf1 v2/v2 mutant mice using primers to amplify exons 6 through 8, exons 7 through 10, and exons 16 and 17. Exons 4-11 are deleted in Rhbdf1 v2/v2 mutant mice; hence no amplicons were generated using either exon 6 forward and exon 8 reverse, or exon 7 forward and exon 10 reverse, primers. However, exon 16 forward and exon 17 reverse primers generated a 211-bp product. d. RNA-Seq analysis of spleens from Rhbdf1 v2/v2 mutant mice indicating loss of exons 4 through 11; however, there is strong evidence for mutant mRNA, as indicated by the presence of the rest of the transcript, which encodes exons 12 through 18 and is not degraded by the nonsense-mediated decay mechanism. e. Schematic representation of exons and introns in the Rhbdf1 v2/v2 mutant allele. 5’ RACE using a gene-specific exon 16-17 fusion primer (GSP) was used to obtain 5’ ends of the Rhbdf1 v2/v2 mutant mRNA. We identified several novel mutant mRNAs with different translation initiation sites that could potentially generate N-terminally truncated RHBDF1 mutant proteins. See supplemental figures for variant protein and 5’ UTR sequences. Alternative exons are indicated as red boxes; predicted translation initiation sites are indicated by “START,” and termination codons are indicated by “STOP.” f. C-terminal Myc-DDK-tagged Rhbdf1 v2/v2 variant protein 1 (lanes 1, 2) or variant protein 2 (lanes 3,4), or empty vector (lanes 5, 6) were transiently expressed in 293T cells, and cell lysates were analyzed using western blotting with FLAG-specific antibody. After visualization of blots with a G:Box chemiluminescent imaging system, blots were washed, blocked in 5% nonfat dry milk, and re-probed with anti-actin antibody. g. Rescue of phenotype in Rhbdf1 −/− MEFs. Rhbdf1 +/+ (top) and Rhbdf1 −/− (bottom) MEFs were transiently transfected with 2 μg of either variant 1 or variant 2 vectors, or an empty vector, using Lipofectamine LTX. 48 h post-transfection, cells were stimulated overnight with either DMSO or 100 nM PMA, and cell-culture supernatants were analyzed using a mouse AREG ELISA kit. Data represent mean ± S.D; *p

    Article Snippet: Briefly, the protocol entails isolation of polyA containing mRNA using oligo-dT magnetic beads, RNA fragmentation, first and second strand cDNA synthesis, ligation of Illumina-specific adapters containing a unique barcode sequence for each library, and PCR amplification.

    Techniques: Functional Assay, Mutagenesis, Mouse Assay, Transgenic Assay, Sequencing, Reverse Transcription Polymerase Chain Reaction, Generated, RNA Sequencing Assay, Variant Assay, Plasmid Preparation, Western Blot, Imaging, Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay

    Flaviviridae infection alters m 6 A modification of RIOK3 and CIRBP mRNA through distinct cellular pathways. (A and B) Coverage plot of MeRIP (color) and input (black) reads in (A) RIOK3 and (B) CIRBP transcripts in Huh7 cells infected with the indicated virus (48 hpi) as determined by MeRIP-seq. Data are representative of three biological replicates. Infection-altered m 6 A peaks (and the number of DRACH motifs within) are indicated in black under the transcript map. (C) (Left) MeRIP-RT-qPCR analysis of relative m 6 A level of RIOK3 and CIRBP in mock- and virus-infected (48 hpi) Huh7 cells. (Right) RNA expression of RIOK3 and CIRBP relative to HPRT1 (right). (D) (Left) MeRIP-RT-qPCR analysis of relative m 6 A level of RIOK3 and CIRBP in mock- and HCV PAMP-transfected (8 h) Huh7 cells. (Right) RNA expression of RIOK3, CIRBP , as well as positive control transcripts IFNB1 and IFIT1 relative to HPRT1 . (E) (Left) MeRIP-RT-qPCR analysis of relative m 6 A level of RIOK3 and CIRBP in mock- and thapsigargin-treated (TG; 16 h) Huh7 cells. (Right) RNA expression of RIOK3, CIRBP , and positive control transcripts HSPA5 and XBP1 relative to HPRT1 . Values are the mean ± SEM of 6 (C-D), 3 (E), or 5 (F) biological replicates. * p

    Journal: bioRxiv

    Article Title: Altered m6A modification of specific cellular transcripts affects Flaviviridae infection

    doi: 10.1101/670984

    Figure Lengend Snippet: Flaviviridae infection alters m 6 A modification of RIOK3 and CIRBP mRNA through distinct cellular pathways. (A and B) Coverage plot of MeRIP (color) and input (black) reads in (A) RIOK3 and (B) CIRBP transcripts in Huh7 cells infected with the indicated virus (48 hpi) as determined by MeRIP-seq. Data are representative of three biological replicates. Infection-altered m 6 A peaks (and the number of DRACH motifs within) are indicated in black under the transcript map. (C) (Left) MeRIP-RT-qPCR analysis of relative m 6 A level of RIOK3 and CIRBP in mock- and virus-infected (48 hpi) Huh7 cells. (Right) RNA expression of RIOK3 and CIRBP relative to HPRT1 (right). (D) (Left) MeRIP-RT-qPCR analysis of relative m 6 A level of RIOK3 and CIRBP in mock- and HCV PAMP-transfected (8 h) Huh7 cells. (Right) RNA expression of RIOK3, CIRBP , as well as positive control transcripts IFNB1 and IFIT1 relative to HPRT1 . (E) (Left) MeRIP-RT-qPCR analysis of relative m 6 A level of RIOK3 and CIRBP in mock- and thapsigargin-treated (TG; 16 h) Huh7 cells. (Right) RNA expression of RIOK3, CIRBP , and positive control transcripts HSPA5 and XBP1 relative to HPRT1 . Values are the mean ± SEM of 6 (C-D), 3 (E), or 5 (F) biological replicates. * p

    Article Snippet: At 48 hours post-infection, total RNA was extracted using TRIzol (Thermo Fisher) and treated with TURBO DNase I (Thermo Fisher). mRNA was purified from 200 µg total RNA from each sample using the Dynabeads mRNA purification kit (Thermo Fisher) and concentrated by ethanol precipitation. mRNA was fragmented using the RNA Fragmentation Reagent (Thermo Fisher) for 15 minutes and purified by ethanol precipitation.

    Techniques: Infection, Modification, Quantitative RT-PCR, RNA Expression, Transfection, Positive Control

    DENV and ZIKV co-opt the RNA binding properties of RRBP1 and vigilin in human cells. a , RRBP1 (left) and vigilin (right) irCLIP reverse transcriptase (RT) stop mapping statistics annotated to the human, DENV, or ZIKV genomes and the ribosomal RNAs (rRNA) from Huh7.5.1 cells infected with MOI of 0.1 for 48 h. b , Histogram of RT stops mapping to the rRNAs from the RRBP1 (top) and vigilin (bottom) irCLIP in uninfected Huh7.5.1 cells. The three cytosolic rRNAs are highlighted. Red dashed line denotes vigilin’s strongest binding site, which is adjacent to RRBP1’s. c , Annotation of peaks called from RRBP1 (top) and vigilin (bottom) irCLIP RT stops mapping to functional elements of human mRNAs including 5’UTR, exons, 3’UTR, and introns. Enrichment values are calculated based on the size of each function domain relative to the human genome. d , RRBP1 (top) and vigilin (bottom) irCLIP RT stops mapped at base resolution to the DENV genome. RT stop intensity was normalized to the total number of unique reads mapping to the viral genome. The 5’UTR and 3’UTR regions are highlighted in red and blue, respectively.

    Journal: Nature microbiology

    Article Title: An RNA-Centric Dissection of Host Complexes Controlling Flavivirus Infection

    doi: 10.1038/s41564-019-0518-2

    Figure Lengend Snippet: DENV and ZIKV co-opt the RNA binding properties of RRBP1 and vigilin in human cells. a , RRBP1 (left) and vigilin (right) irCLIP reverse transcriptase (RT) stop mapping statistics annotated to the human, DENV, or ZIKV genomes and the ribosomal RNAs (rRNA) from Huh7.5.1 cells infected with MOI of 0.1 for 48 h. b , Histogram of RT stops mapping to the rRNAs from the RRBP1 (top) and vigilin (bottom) irCLIP in uninfected Huh7.5.1 cells. The three cytosolic rRNAs are highlighted. Red dashed line denotes vigilin’s strongest binding site, which is adjacent to RRBP1’s. c , Annotation of peaks called from RRBP1 (top) and vigilin (bottom) irCLIP RT stops mapping to functional elements of human mRNAs including 5’UTR, exons, 3’UTR, and introns. Enrichment values are calculated based on the size of each function domain relative to the human genome. d , RRBP1 (top) and vigilin (bottom) irCLIP RT stops mapped at base resolution to the DENV genome. RT stop intensity was normalized to the total number of unique reads mapping to the viral genome. The 5’UTR and 3’UTR regions are highlighted in red and blue, respectively.

    Article Snippet: Ribosomal RNA was depleted using the RiboMinus Transcriptome Isolation Kit, human/mouse (Thermo Fisher Scientific) as per the manufactures instructions starting with 5 μg of total RNA per sample. rRNA-depleted samples were fragmented using the RNA Fragmentation Reagent (Thermo Fisher Scientific) at 90°C for 30 seconds.

    Techniques: RNA Binding Assay, Infection, Binding Assay, Functional Assay