rna clean  (Zymo Research)


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    RNA Clean & Concentrator-5 (DNase Included)
    Description:
    The RNA Clean & Concentrator kits provide a simple and reliable method for the rapid preparation of high-quality RT-PCR-ready, DNA-free (R1013, R1014) RNA. This simple procedure is based on the use of a unique single-buffer system and Zymo-Spin column technology that allows for selective recovery of total RNA (> 17 nt), large RNAs (> 200 nt), and/or small RNAs (17-200 nt). The procedure is easy: Add binding buffer and ethanol to your sample, then bind, wash and elute ultra pure RNA. The RNA can be eluted from the Zymo-Spin IC Column in as little as ≥ 6 µl of RNase-free water. The highly-concentrated, purified RNA is suitable for all subsequent analyses and molecular manipulations.
    Catalog Number:
    R1013
    Price:
    None
    Applications:
    RNA Purification
    Size:
    50 units
    Category:
    Life Science Reagents and Media
    Score:
    85
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    Structured Review

    Zymo Research rna clean
    RNA Clean & Concentrator-5 (DNase Included)
    The RNA Clean & Concentrator kits provide a simple and reliable method for the rapid preparation of high-quality RT-PCR-ready, DNA-free (R1013, R1014) RNA. This simple procedure is based on the use of a unique single-buffer system and Zymo-Spin column technology that allows for selective recovery of total RNA (> 17 nt), large RNAs (> 200 nt), and/or small RNAs (17-200 nt). The procedure is easy: Add binding buffer and ethanol to your sample, then bind, wash and elute ultra pure RNA. The RNA can be eluted from the Zymo-Spin IC Column in as little as ≥ 6 µl of RNase-free water. The highly-concentrated, purified RNA is suitable for all subsequent analyses and molecular manipulations.
    https://www.bioz.com/result/rna clean/product/Zymo Research
    Average 99 stars, based on 57 article reviews
    Price from $9.99 to $1999.99
    rna clean - by Bioz Stars, 2019-10
    99/100 stars

    Images

    1) Product Images from "Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis"

    Article Title: Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis

    Journal: eLife

    doi: 10.7554/eLife.09591

    Sequence relationships between Pol IV/RDR2-dependent RNAs (P4R2 RNAs) and small interfering RNA (siRNAs). ( A ) Correspondence between P4R2 RNA and siRNA loci. P4R2 RNAs were mapped to the Arabidopsis reference genome (TAIR10) and the frequency at which 24 nt siRNAs overlap these P4R2 genomic positions was calculated. To be considered for this analysis, specific siRNAs had to be represented by at least five reads in wild-type Col-0. Supplementary file 2 shows that P4R2 RNA loci include loci confirmed by Li et al., 2015 to generate Pol IV-dependent transcripts. ( B ) P4R2 RNA and 24 nt siRNA spatial relationships. The top panel shows the frequency distribution of P4R2 RNA 5’ end positions relative to siRNA 5’ ends. At position zero on the x-axis, P4R2 RNAs and siRNAs share the same 5’ terminus. Negative values indicate how far (in nucleotides) the 5’ end of the P4R2 RNA is located upstream of an siRNA start position. Likewise, positive values indicate how far the 5’ end of a P4R2 RNA is located downstream of an siRNA start position. The lower panel shows the frequency with which P4R2 RNAs and siRNAs align at 3’ ends. At position zero on the x-axis, P4R2 RNAs and siRNAs share the same 3’ terminus. Negative values occur when P4R2 RNAs end upstream of siRNA 3’ ends, and positive values occur when P4R2 RNAs end downstream of siRNA 3’ ends (computed using FEATnotator, v1.2.2, Podicheti and Mockaitis, 2015 ). DOI: http://dx.doi.org/10.7554/eLife.09591.012
    Figure Legend Snippet: Sequence relationships between Pol IV/RDR2-dependent RNAs (P4R2 RNAs) and small interfering RNA (siRNAs). ( A ) Correspondence between P4R2 RNA and siRNA loci. P4R2 RNAs were mapped to the Arabidopsis reference genome (TAIR10) and the frequency at which 24 nt siRNAs overlap these P4R2 genomic positions was calculated. To be considered for this analysis, specific siRNAs had to be represented by at least five reads in wild-type Col-0. Supplementary file 2 shows that P4R2 RNA loci include loci confirmed by Li et al., 2015 to generate Pol IV-dependent transcripts. ( B ) P4R2 RNA and 24 nt siRNA spatial relationships. The top panel shows the frequency distribution of P4R2 RNA 5’ end positions relative to siRNA 5’ ends. At position zero on the x-axis, P4R2 RNAs and siRNAs share the same 5’ terminus. Negative values indicate how far (in nucleotides) the 5’ end of the P4R2 RNA is located upstream of an siRNA start position. Likewise, positive values indicate how far the 5’ end of a P4R2 RNA is located downstream of an siRNA start position. The lower panel shows the frequency with which P4R2 RNAs and siRNAs align at 3’ ends. At position zero on the x-axis, P4R2 RNAs and siRNAs share the same 3’ terminus. Negative values occur when P4R2 RNAs end upstream of siRNA 3’ ends, and positive values occur when P4R2 RNAs end downstream of siRNA 3’ ends (computed using FEATnotator, v1.2.2, Podicheti and Mockaitis, 2015 ). DOI: http://dx.doi.org/10.7554/eLife.09591.012

    Techniques Used: Sequencing, Small Interfering RNA

    Pol IV transcripts generated in vitro share features of Pol IV/RDR2-dependent RNAs (P4R2 RNAs) in vivo. ( A and B ) Size and frequency of RNAs transcribed by polymerase (Pol) IV or II in vitro. Pol IV and Pol II were affinity purified by virtue of FLAG epitope tags fused to the C-termini of the NRPD1 or NRPB2 subunits, respectively. In the case of Pol IV, the transgenic NRPD1-FLAG line is null for the endogenous NRPD1 and RDR2 genes, such that Pol IV is free of associated RDR2. Transcripts generated using closed-circular single-stranded M13 virus as the DNA template were subjected to RNA-seq. The frequency and sizes of mapped reads are plotted. ( C and D ) Sequence logos for the 5’ and 3’ ends of Pol IV and II in vitro transcripts. RNA-seq, RNA sequencing. DOI: http://dx.doi.org/10.7554/eLife.09591.015
    Figure Legend Snippet: Pol IV transcripts generated in vitro share features of Pol IV/RDR2-dependent RNAs (P4R2 RNAs) in vivo. ( A and B ) Size and frequency of RNAs transcribed by polymerase (Pol) IV or II in vitro. Pol IV and Pol II were affinity purified by virtue of FLAG epitope tags fused to the C-termini of the NRPD1 or NRPB2 subunits, respectively. In the case of Pol IV, the transgenic NRPD1-FLAG line is null for the endogenous NRPD1 and RDR2 genes, such that Pol IV is free of associated RDR2. Transcripts generated using closed-circular single-stranded M13 virus as the DNA template were subjected to RNA-seq. The frequency and sizes of mapped reads are plotted. ( C and D ) Sequence logos for the 5’ and 3’ ends of Pol IV and II in vitro transcripts. RNA-seq, RNA sequencing. DOI: http://dx.doi.org/10.7554/eLife.09591.015

    Techniques Used: Generated, In Vitro, In Vivo, Affinity Purification, FLAG-tag, Transgenic Assay, RNA Sequencing Assay, Sequencing

    3’ mismatches detected in 24 nt siRNAs and Pol IV/RDR2-dependent RNAs (P4R2 RNAs) may reflect RDR2 terminal transferase activity. ( A ) Genome browser view of P4R2 RNAs (shades of blue) and 24 nt small interfering RNA (siRNAs) (shades of gray) at a representative locus, an AtSN1 retrotransposon on chromosome 3. Each horizontal bar represents a specific RNA sequence (RNA-seq), with arrows depicting their direction relative to the Arabidopsis reference genome sequence (TAIR10). The intensity of shading reflects the abundance of each RNA species in the RNA-seq dataset. Brightly colored nucleotides, color coded for A, G, C, or U (see inset), represent nucleotides that do not match the corresponding DNA sequence of the locus. The dotted line highlights the coincident 5’ ends of the most abundant P4R2 RNAs at the locus (colored deep purple) and the most abundant siRNAs (colored black). ( B ) Heat map depicting the frequency of mismatched nucleotides at each position of RNAs ranging in size from 15 to 76 nt in dcl2 dcl3 dcl4 triple mutant plants. To correct for the frequency of errors inherent to sequencing, mismatch values for each position of 15–76 nt RNAs in wild-type plants were subtracted prior to plotting the data. Only read sequences with single mismatches or perfect matches to the reference genome were utilized for this analysis. ( C ) Over-expression and purification of recombinant RDR2. The image on the left shows a 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) gel, stained with Coomassie blue, showing molecular weight markers (M), proteins of un-infected High Five cells (lane 1), proteins of High Five cells 72 hr after infection with baculovirus expressing recombinant RNA-dependent RNA polymerase 2 (RDR2) (lane 2), and purified recombinant V5-tagged RDR2 after affinity purification and elution with V5 peptide. The image at right shows anti-RDR2 and anti-V5 immunoblots of the same three protein samples. For RDR2 detection, rabbit anti-RDR2 primary antibody was used in conjunction with donkey anti-rabbit HRP-conjugated secondary antibody. Detection of V5-tagged RDR2 involved anti-V5 HRP conjugate antibody. ( D ) RDR2 terminal transferase activity. Recombinant RDR2 or an active-site mutant form of RDR2 (RDR2-ASM) was incubated with alpha-labeled 32 P-CTP and 51 nt RNA substrates bearing 3’ hydroxyl or 3’ dideoxy termini. Reaction products were subjected to denaturing polyacrylamide gel electrophoresis (PAGE) and autoradiography. For gel lane 4, reaction products were treated with RNase One, which degrades single-stranded RNAs, prior to PAGE. RNA size markers were run in lane M. The 51 nt RNA template, 5’ end-labeled using T4 polynucleotide kinase, was run as a size marker in the lane at far right. DOI: http://dx.doi.org/10.7554/eLife.09591.016
    Figure Legend Snippet: 3’ mismatches detected in 24 nt siRNAs and Pol IV/RDR2-dependent RNAs (P4R2 RNAs) may reflect RDR2 terminal transferase activity. ( A ) Genome browser view of P4R2 RNAs (shades of blue) and 24 nt small interfering RNA (siRNAs) (shades of gray) at a representative locus, an AtSN1 retrotransposon on chromosome 3. Each horizontal bar represents a specific RNA sequence (RNA-seq), with arrows depicting their direction relative to the Arabidopsis reference genome sequence (TAIR10). The intensity of shading reflects the abundance of each RNA species in the RNA-seq dataset. Brightly colored nucleotides, color coded for A, G, C, or U (see inset), represent nucleotides that do not match the corresponding DNA sequence of the locus. The dotted line highlights the coincident 5’ ends of the most abundant P4R2 RNAs at the locus (colored deep purple) and the most abundant siRNAs (colored black). ( B ) Heat map depicting the frequency of mismatched nucleotides at each position of RNAs ranging in size from 15 to 76 nt in dcl2 dcl3 dcl4 triple mutant plants. To correct for the frequency of errors inherent to sequencing, mismatch values for each position of 15–76 nt RNAs in wild-type plants were subtracted prior to plotting the data. Only read sequences with single mismatches or perfect matches to the reference genome were utilized for this analysis. ( C ) Over-expression and purification of recombinant RDR2. The image on the left shows a 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) gel, stained with Coomassie blue, showing molecular weight markers (M), proteins of un-infected High Five cells (lane 1), proteins of High Five cells 72 hr after infection with baculovirus expressing recombinant RNA-dependent RNA polymerase 2 (RDR2) (lane 2), and purified recombinant V5-tagged RDR2 after affinity purification and elution with V5 peptide. The image at right shows anti-RDR2 and anti-V5 immunoblots of the same three protein samples. For RDR2 detection, rabbit anti-RDR2 primary antibody was used in conjunction with donkey anti-rabbit HRP-conjugated secondary antibody. Detection of V5-tagged RDR2 involved anti-V5 HRP conjugate antibody. ( D ) RDR2 terminal transferase activity. Recombinant RDR2 or an active-site mutant form of RDR2 (RDR2-ASM) was incubated with alpha-labeled 32 P-CTP and 51 nt RNA substrates bearing 3’ hydroxyl or 3’ dideoxy termini. Reaction products were subjected to denaturing polyacrylamide gel electrophoresis (PAGE) and autoradiography. For gel lane 4, reaction products were treated with RNase One, which degrades single-stranded RNAs, prior to PAGE. RNA size markers were run in lane M. The 51 nt RNA template, 5’ end-labeled using T4 polynucleotide kinase, was run as a size marker in the lane at far right. DOI: http://dx.doi.org/10.7554/eLife.09591.016

    Techniques Used: Activity Assay, Small Interfering RNA, Sequencing, RNA Sequencing Assay, Mutagenesis, Over Expression, Purification, Recombinant, Electrophoresis, Polyacrylamide Gel Electrophoresis, Staining, Molecular Weight, Infection, Expressing, Affinity Purification, Western Blot, Incubation, Labeling, Autoradiography, Marker

    RNA blot analyses of 24 nt siRNAs and their precursors. ( A ) The small RNA blot was successively hybridized to probes representing either strand of the siR1003 duplex, a small interfering RNA (siRNA) that is derived from intergenic regions separating 5S ribosomal RNA (rRNA) gene repeats (top two images), as well as to a trans -acting siRNA (ta-siR255) and a microRNA (miR160) probe. An image of the stained gel under fluorescent illumination (in the region that includes 5S rRNA and transfer RNAs [tRNAs]) is shown at the bottom as a loading control. ( B ) RNA blot of small RNAs isolated from wild-type (ecotype Col-0) or dcl2 dcl3 dcl4 triple mutant ( dcl2/3/4 ) plants, with or without prior treatment with ribonuclease V1 or ribonuclease A. The blot was hybridized to a probe designed to detect siR1003 ‘sense’, which arises from 5S rRNA gene intergenic spacers. ( C ) Dicing of precursor RNAs by DICER-like 3 (DCL3) in vitro. RNA isolated from wild-type (ecotype Col-0) or from dcl2/3/4 triple mutant plants was incubated with anti-FLAG resin that had been incubated with protein extraction buffer, a cell-free extract of wild-type (Col-0) plants, or a cell-free extract of transgenic plants expressing FLAG-tagged DCL3. RNAs were then purified, subjected to blotting and hybridized to the siR1003 ‘sense’ probe. DOI: http://dx.doi.org/10.7554/eLife.09591.004
    Figure Legend Snippet: RNA blot analyses of 24 nt siRNAs and their precursors. ( A ) The small RNA blot was successively hybridized to probes representing either strand of the siR1003 duplex, a small interfering RNA (siRNA) that is derived from intergenic regions separating 5S ribosomal RNA (rRNA) gene repeats (top two images), as well as to a trans -acting siRNA (ta-siR255) and a microRNA (miR160) probe. An image of the stained gel under fluorescent illumination (in the region that includes 5S rRNA and transfer RNAs [tRNAs]) is shown at the bottom as a loading control. ( B ) RNA blot of small RNAs isolated from wild-type (ecotype Col-0) or dcl2 dcl3 dcl4 triple mutant ( dcl2/3/4 ) plants, with or without prior treatment with ribonuclease V1 or ribonuclease A. The blot was hybridized to a probe designed to detect siR1003 ‘sense’, which arises from 5S rRNA gene intergenic spacers. ( C ) Dicing of precursor RNAs by DICER-like 3 (DCL3) in vitro. RNA isolated from wild-type (ecotype Col-0) or from dcl2/3/4 triple mutant plants was incubated with anti-FLAG resin that had been incubated with protein extraction buffer, a cell-free extract of wild-type (Col-0) plants, or a cell-free extract of transgenic plants expressing FLAG-tagged DCL3. RNAs were then purified, subjected to blotting and hybridized to the siR1003 ‘sense’ probe. DOI: http://dx.doi.org/10.7554/eLife.09591.004

    Techniques Used: Northern blot, Small Interfering RNA, Derivative Assay, Staining, Isolation, Mutagenesis, In Vitro, Incubation, Protein Extraction, Transgenic Assay, Expressing, Purification

    P4R2 RNAs are co-dependent on Pol IV and RDR2. ( A–C ) Browser views of three additional 24 nt small interfering RNA (siRNA) loci at which Pol IV/RDR2-dependent RNAs (P4R2 RNAs) that accumulate in dcl2 dcl3 dcl4 ( dcl2/3/4 ) mutants are also observed in wild-type (Col-0) plants (with five or more reads for at least one P4R2 species) but are not observed in nrpd1 polymerase IV (Pol IV) or rdr2 mutants. DOI: http://dx.doi.org/10.7554/eLife.09591.011
    Figure Legend Snippet: P4R2 RNAs are co-dependent on Pol IV and RDR2. ( A–C ) Browser views of three additional 24 nt small interfering RNA (siRNA) loci at which Pol IV/RDR2-dependent RNAs (P4R2 RNAs) that accumulate in dcl2 dcl3 dcl4 ( dcl2/3/4 ) mutants are also observed in wild-type (Col-0) plants (with five or more reads for at least one P4R2 species) but are not observed in nrpd1 polymerase IV (Pol IV) or rdr2 mutants. DOI: http://dx.doi.org/10.7554/eLife.09591.011

    Techniques Used: Small Interfering RNA

    Browser view of Pol IV/RDR2-dependent RNAs (P4R2 RNAs) and 24/23 nt small interfering RNA (siRNAs) in the intergenic spacer region of a 5S ribosomal RNA (rRNA) gene repeat unit. An isolated 5S rRNA gene repeat (∼500 bp, gray horizontal bar with red transcript region) is shown within its 5 kb chromosomal context, flanked by two transposable elements, shown in yellow, and a pseudogene, shown in blue. Below the diagram, P4R2 RNAs are depicted as horizontal bars shown in shades of blue whereas 24 and 23 nt siRNAs are shown in shades of gray to black, with color intensity reflecting abundance (read counts are provided for several examples). Each bar represents a specific RNA sequence, with arrows depicting the RNA strand orientation relative to the reference genome sequence (TAIR10). Dotted vertical lines provide alignments and show that the ends of highly abundant ( > 100 reads) siRNA species tend to coincide with the ends of P4R2 RNAs for which there is more than a single read. DOI: http://dx.doi.org/10.7554/eLife.09591.009
    Figure Legend Snippet: Browser view of Pol IV/RDR2-dependent RNAs (P4R2 RNAs) and 24/23 nt small interfering RNA (siRNAs) in the intergenic spacer region of a 5S ribosomal RNA (rRNA) gene repeat unit. An isolated 5S rRNA gene repeat (∼500 bp, gray horizontal bar with red transcript region) is shown within its 5 kb chromosomal context, flanked by two transposable elements, shown in yellow, and a pseudogene, shown in blue. Below the diagram, P4R2 RNAs are depicted as horizontal bars shown in shades of blue whereas 24 and 23 nt siRNAs are shown in shades of gray to black, with color intensity reflecting abundance (read counts are provided for several examples). Each bar represents a specific RNA sequence, with arrows depicting the RNA strand orientation relative to the reference genome sequence (TAIR10). Dotted vertical lines provide alignments and show that the ends of highly abundant ( > 100 reads) siRNA species tend to coincide with the ends of P4R2 RNAs for which there is more than a single read. DOI: http://dx.doi.org/10.7554/eLife.09591.009

    Techniques Used: Small Interfering RNA, Isolation, Sequencing

    Number of unique sequences among RNAs of 15–94 nt in wild-type or dcl2 dcl3 dcl4 ( dcl2/3/4/) triple mutants. RNAs representing a specific sequence and a particular strand polarity were counted only once, regardless of the actual abundance of RNA sequencing (RNA-seq) reads for that sequence in the dataset. RNA-seq counts for wild-type are shown in blue. RNA-seq counts for dcl2 dcl3 dcl4 mutants are shown in orange. Overlapping read counts are shown in deep red. DOI: http://dx.doi.org/10.7554/eLife.09591.008
    Figure Legend Snippet: Number of unique sequences among RNAs of 15–94 nt in wild-type or dcl2 dcl3 dcl4 ( dcl2/3/4/) triple mutants. RNAs representing a specific sequence and a particular strand polarity were counted only once, regardless of the actual abundance of RNA sequencing (RNA-seq) reads for that sequence in the dataset. RNA-seq counts for wild-type are shown in blue. RNA-seq counts for dcl2 dcl3 dcl4 mutants are shown in orange. Overlapping read counts are shown in deep red. DOI: http://dx.doi.org/10.7554/eLife.09591.008

    Techniques Used: Sequencing, RNA Sequencing Assay

    Biogenesis of 24 nt siRNAs and their role in RNA-directed DNA methylation. A simplified cartoon of the RNA-directed DNA methylation pathway. Polymerase (Pol) IV and RNA-dependent RNA polymerase (RDR2) physically associate and are required for the synthesis of double-stranded RNAs (dsRNA) that are diced by DICER-like 3 (DCL3) into 24 nt siRNA duplexes. Upon loading into Argonaute 4 (AGO4), the siRNA-AGO4 complex finds its target sites by binding to Pol V transcripts and by interacting with the C-terminal domain (CTD) of the Pol V largest subunit. The cytosine methyltransferase DRM2 is ultimately recruited to Pol V-transcribed loci, resulting in de novo cytosine methylation in all sequence contexts (CG, CHG and CHH; where H represents a nucleotide other than G). DOI: http://dx.doi.org/10.7554/eLife.09591.003
    Figure Legend Snippet: Biogenesis of 24 nt siRNAs and their role in RNA-directed DNA methylation. A simplified cartoon of the RNA-directed DNA methylation pathway. Polymerase (Pol) IV and RNA-dependent RNA polymerase (RDR2) physically associate and are required for the synthesis of double-stranded RNAs (dsRNA) that are diced by DICER-like 3 (DCL3) into 24 nt siRNA duplexes. Upon loading into Argonaute 4 (AGO4), the siRNA-AGO4 complex finds its target sites by binding to Pol V transcripts and by interacting with the C-terminal domain (CTD) of the Pol V largest subunit. The cytosine methyltransferase DRM2 is ultimately recruited to Pol V-transcribed loci, resulting in de novo cytosine methylation in all sequence contexts (CG, CHG and CHH; where H represents a nucleotide other than G). DOI: http://dx.doi.org/10.7554/eLife.09591.003

    Techniques Used: DNA Methylation Assay, Binding Assay, Methylation, Sequencing

    2) Product Images from "The LINK-A lncRNA interacts with PI(3,4,5)P3 to hyperactivate AKT and confer resistance to AKT inhibitors"

    Article Title: The LINK-A lncRNA interacts with PI(3,4,5)P3 to hyperactivate AKT and confer resistance to AKT inhibitors

    Journal: Nature cell biology

    doi: 10.1038/ncb3473

    Functional involvement of LINK-A -PIP 3 interaction in mediating AKT activation ( a and b ) Detection of PIP 3 (a) and phospho-AKT (b) level in DLD-1 PIK3CA +/+ or PIK3CA −/− cells with or without EGF stimulation. ( c ) Schematic illustration of intracellular delivery of PIP 3 and/or LINK-A to DLD-1 cells. ( d and e ) RT-qPCR determination of LINK-A copy number (d) or IB detection of indicated proteins (e) in DLD-1 PIK3CA +/+ cells delivered with indicated RNA transcripts with or without EGF stimulation. ( f–h ) PIP 3 mass ELISA (f), RT-qPCR determination of LINK-A copy number (g) or IB detection of indicated proteins (h) in DLD-1 PIK3CA +/− cells delivered with PIP 3 and/or LINK-A transcripts with or without EGF stimulation. ( i–k ) PIP 3 mass ELISA (i), RT-qPCR determination of LINK-A copy number (j) or IB detection of indicated proteins (k) in DLD-1 PIK3CA +/− cells delivered with PIP 3 and indicated LINK-A deletion transcripts with or without EGF stimulation. ( l ) IB detection of indicated proteins in DLD-1 PIK3CA +/− cells delivered with PIP 3 and indicated LINK-A single nucleotide mutated transcripts with or without EGF stimulation. ( m and n ) IB detection of immunoprecipitated AKT (m) and quantification of AKT-associated PIP 3 (n) in DLD-1 PIK3CA +/+ cells delivered with indicated LINK-A single nucleotide mutated transcripts with or without EGF stimulation. For a, d, f, g, i, j and n , mean ± s.e.m. were derived from n =3 independent experiments (n.s. p > 0.05, * p
    Figure Legend Snippet: Functional involvement of LINK-A -PIP 3 interaction in mediating AKT activation ( a and b ) Detection of PIP 3 (a) and phospho-AKT (b) level in DLD-1 PIK3CA +/+ or PIK3CA −/− cells with or without EGF stimulation. ( c ) Schematic illustration of intracellular delivery of PIP 3 and/or LINK-A to DLD-1 cells. ( d and e ) RT-qPCR determination of LINK-A copy number (d) or IB detection of indicated proteins (e) in DLD-1 PIK3CA +/+ cells delivered with indicated RNA transcripts with or without EGF stimulation. ( f–h ) PIP 3 mass ELISA (f), RT-qPCR determination of LINK-A copy number (g) or IB detection of indicated proteins (h) in DLD-1 PIK3CA +/− cells delivered with PIP 3 and/or LINK-A transcripts with or without EGF stimulation. ( i–k ) PIP 3 mass ELISA (i), RT-qPCR determination of LINK-A copy number (j) or IB detection of indicated proteins (k) in DLD-1 PIK3CA +/− cells delivered with PIP 3 and indicated LINK-A deletion transcripts with or without EGF stimulation. ( l ) IB detection of indicated proteins in DLD-1 PIK3CA +/− cells delivered with PIP 3 and indicated LINK-A single nucleotide mutated transcripts with or without EGF stimulation. ( m and n ) IB detection of immunoprecipitated AKT (m) and quantification of AKT-associated PIP 3 (n) in DLD-1 PIK3CA +/+ cells delivered with indicated LINK-A single nucleotide mutated transcripts with or without EGF stimulation. For a, d, f, g, i, j and n , mean ± s.e.m. were derived from n =3 independent experiments (n.s. p > 0.05, * p

    Techniques Used: Functional Assay, Activation Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Derivative Assay

    Global identification of lipid-interacting lncRNAs and characterization of LINK-A -lipid interaction ( a ) Experimental scheme for identification of lipid-bound lncRNAs in triple-negative breast cancer (TNBC). ( b ) Pie chart of percentage of TNBC-upregulated lncRNAs that associated with cellular lipids. ( c ) Scatter blot representing lipid enrichment of top 9 lncRNAs in normal breast tissue (N) or malignant breast cancer (T). X and Y axis represent log2 scale of normalized lncRNA density (Lipid/Total). ( d ) Heatmap of lipid enrichment of top 9 lncRNAs based on lipid-RNA pulldown followed by RT-qPCR. Red/blue indicates increased/decreased fold change in lipid enrichment over control beads. ( e ) RNA-lipid overlay assay showing binding of LINK-A to PC and PIP 3 . In vitro transcribed biotinylated RNA transcripts, as indicated, were applied to membrane lipid strips. ( f ) Upper panel: graphic illustration of the PIP 3 - LINK-A interaction detected by FRET assay. Lower panel: fluorescence spectra of BODIPY FL-PIP 3 (donor) in the presence of Alexa-555-Strep (blue) or Alexa-555-Strep-biotin- LINK-A (red; λexc = 475 nm). ( g ) Representative fluorescence spectra of BODIPY FL-PIP 3 upon titration of increasing concentrations of LINK-A (0 ~ 400 nM; λexc = 490 nm). ( h ) Fitting the fluorescence quenching of BODIPY FL-PIP 3 induced by LINK-A with one site binding equation. Data fitting yielded a dissociation constant (K d ) of 112 ± 37 nM (mean ± s.e.m. were derived from n =3 independent experiments). ( i ) In vitro RNA-lipid binding using in vitro transcribed biotinylated LINK-A sense or antisense, and lipid-coated beads followed by dot-blot assays (upper panel). Bottom panel: graphic illustration of oligonucleotides base-pairing LINK-A sequence. ( j ) Upper panel: graphic illustration of LINK-A -PC/PIP 3 binding. Lower panel: RNA-lipid overlay assay showing the binding of full-length LINK-A and PC- or PIP 3 -binding region deletion transcripts (ΔPC and ΔPIP 3 , respectively).
    Figure Legend Snippet: Global identification of lipid-interacting lncRNAs and characterization of LINK-A -lipid interaction ( a ) Experimental scheme for identification of lipid-bound lncRNAs in triple-negative breast cancer (TNBC). ( b ) Pie chart of percentage of TNBC-upregulated lncRNAs that associated with cellular lipids. ( c ) Scatter blot representing lipid enrichment of top 9 lncRNAs in normal breast tissue (N) or malignant breast cancer (T). X and Y axis represent log2 scale of normalized lncRNA density (Lipid/Total). ( d ) Heatmap of lipid enrichment of top 9 lncRNAs based on lipid-RNA pulldown followed by RT-qPCR. Red/blue indicates increased/decreased fold change in lipid enrichment over control beads. ( e ) RNA-lipid overlay assay showing binding of LINK-A to PC and PIP 3 . In vitro transcribed biotinylated RNA transcripts, as indicated, were applied to membrane lipid strips. ( f ) Upper panel: graphic illustration of the PIP 3 - LINK-A interaction detected by FRET assay. Lower panel: fluorescence spectra of BODIPY FL-PIP 3 (donor) in the presence of Alexa-555-Strep (blue) or Alexa-555-Strep-biotin- LINK-A (red; λexc = 475 nm). ( g ) Representative fluorescence spectra of BODIPY FL-PIP 3 upon titration of increasing concentrations of LINK-A (0 ~ 400 nM; λexc = 490 nm). ( h ) Fitting the fluorescence quenching of BODIPY FL-PIP 3 induced by LINK-A with one site binding equation. Data fitting yielded a dissociation constant (K d ) of 112 ± 37 nM (mean ± s.e.m. were derived from n =3 independent experiments). ( i ) In vitro RNA-lipid binding using in vitro transcribed biotinylated LINK-A sense or antisense, and lipid-coated beads followed by dot-blot assays (upper panel). Bottom panel: graphic illustration of oligonucleotides base-pairing LINK-A sequence. ( j ) Upper panel: graphic illustration of LINK-A -PC/PIP 3 binding. Lower panel: RNA-lipid overlay assay showing the binding of full-length LINK-A and PC- or PIP 3 -binding region deletion transcripts (ΔPC and ΔPIP 3 , respectively).

    Techniques Used: Quantitative RT-PCR, Overlay Assay, Binding Assay, In Vitro, Fluorescence, Titration, Derivative Assay, Dot Blot, Sequencing

    3) Product Images from "Comprehensive RNA-Seq transcriptomic profiling across 11 organs, 4 ages, and 2 sexes of Fischer 344 rats"

    Article Title: Comprehensive RNA-Seq transcriptomic profiling across 11 organs, 4 ages, and 2 sexes of Fischer 344 rats

    Journal: Scientific Data

    doi: 10.1038/sdata.2014.13

    Quality assessment metrics for RNA-Seq data. Box plots representing ( a ) GC content (%) and ( b ) Phred quality score distribution over all reads across all samples in each base (i.e., sequencing cycle). The box and horizontal bar represent the interquartile range and median of the ( a ) GC content and ( b ) median of Phred quality score over all reads. ( c ) Box plot representing the percentage of reads ( y -axis) that appear N times ( x -axis) relative to the number of unique reads from each sequencing sample across all samples. ( d ) The percentage of reads mapped (ratio, Mean ±s.e., n =32 in normal organs or 16 in sexual organs) to genomic regions, AceView exons, ERCCs and rRNA in each organ.
    Figure Legend Snippet: Quality assessment metrics for RNA-Seq data. Box plots representing ( a ) GC content (%) and ( b ) Phred quality score distribution over all reads across all samples in each base (i.e., sequencing cycle). The box and horizontal bar represent the interquartile range and median of the ( a ) GC content and ( b ) median of Phred quality score over all reads. ( c ) Box plot representing the percentage of reads ( y -axis) that appear N times ( x -axis) relative to the number of unique reads from each sequencing sample across all samples. ( d ) The percentage of reads mapped (ratio, Mean ±s.e., n =32 in normal organs or 16 in sexual organs) to genomic regions, AceView exons, ERCCs and rRNA in each organ.

    Techniques Used: RNA Sequencing Assay, Gas Chromatography, Sequencing

    4) Product Images from "In vivo cleavage rules and target repertoire of RNase III in Escherichia coli"

    Article Title: In vivo cleavage rules and target repertoire of RNase III in Escherichia coli

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky684

    Classification of RNase III cleavage sites. ( A ). Distribution of cleavage sites over genomic elements compared to the background distribution. Positions along the genome were classified following EcoCyc ( 27 ), ( http://ecocyc.org ) version 20.0 into seven categories: 5UTR (5′UTR); CDS; 3UTR (3′UTR); AS (unannotated A nti S ense); IGR ( I nter G enic R egion); IGT ( I nter G enic within T ranscript) and ncRNA (all annotated non-coding RNAs including small RNA, tRNA and annotated cis antisense). Of note, except for cleavage sites in tRNAs residing in rRNA operons, no cleavage site was annotated as tRNA. Cleavage sites in the vicinity of tRNAs were detected, however, in regions annotated as IGRs. See Supplementary Data for additional annotation details. Red—distribution of genomic elements where RNase III cleavage sites were identified; black—background distribution (counted over all genomic positions). ( B ) Distribution of number of cleavage sites per target.
    Figure Legend Snippet: Classification of RNase III cleavage sites. ( A ). Distribution of cleavage sites over genomic elements compared to the background distribution. Positions along the genome were classified following EcoCyc ( 27 ), ( http://ecocyc.org ) version 20.0 into seven categories: 5UTR (5′UTR); CDS; 3UTR (3′UTR); AS (unannotated A nti S ense); IGR ( I nter G enic R egion); IGT ( I nter G enic within T ranscript) and ncRNA (all annotated non-coding RNAs including small RNA, tRNA and annotated cis antisense). Of note, except for cleavage sites in tRNAs residing in rRNA operons, no cleavage site was annotated as tRNA. Cleavage sites in the vicinity of tRNAs were detected, however, in regions annotated as IGRs. See Supplementary Data for additional annotation details. Red—distribution of genomic elements where RNase III cleavage sites were identified; black—background distribution (counted over all genomic positions). ( B ) Distribution of number of cleavage sites per target.

    Techniques Used:

    5) Product Images from "The RNA Complement of Outer Membrane Vesicles From Salmonella enterica Serovar Typhimurium Under Distinct Culture Conditions"

    Article Title: The RNA Complement of Outer Membrane Vesicles From Salmonella enterica Serovar Typhimurium Under Distinct Culture Conditions

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.02015

    Relative composition of the OMVs RNA cargo for each culture condition. Individual colored bars represent the relative amount of each RNA class in the respective OMV RNA-Seq data (average of the biological triplicates). Number of reads have been normalized with DESeq2 . RNA classes are defined from the Salmonella LT2 genome annotation (NCBI accession number AE006468.2 ) or pSLT plasmid annotation (NCBI accession number AE006471.2 ), with supplemental small RNA annotations from Srikumar et al. (2015) . The existence of significant proportion differences among conditions for a specific RNA class was assessed by one-way ANOVA and F -test. Significant differences for rRNA and mRNA classes between conditions are indicated under the graph, determined by Tukey’s range test. Further details for other RNA classes can be found in Supplementary Dataset B .
    Figure Legend Snippet: Relative composition of the OMVs RNA cargo for each culture condition. Individual colored bars represent the relative amount of each RNA class in the respective OMV RNA-Seq data (average of the biological triplicates). Number of reads have been normalized with DESeq2 . RNA classes are defined from the Salmonella LT2 genome annotation (NCBI accession number AE006468.2 ) or pSLT plasmid annotation (NCBI accession number AE006471.2 ), with supplemental small RNA annotations from Srikumar et al. (2015) . The existence of significant proportion differences among conditions for a specific RNA class was assessed by one-way ANOVA and F -test. Significant differences for rRNA and mRNA classes between conditions are indicated under the graph, determined by Tukey’s range test. Further details for other RNA classes can be found in Supplementary Dataset B .

    Techniques Used: RNA Sequencing Assay, Plasmid Preparation

    Repartition of enriched RNAs in/on OMVs isolated from the different analyzed in vitro culture conditions. UpSet plot representing the number of enriched RNAs shared between different conditions. One unit represents one expressed gene. The graph was generated using the UpSet R package. Enriched RNAs were determined using DESeq2 , calculating differential presence between intracellular and OMV-associated fractions for each condition. Expressed genes with an average number of reads over 5 for a given set of triplicates, a log 2 Fold Change over 2, and a corresponding adjusted p -value under 0.05 were selected. ncRNA class attribution is defined from the Salmonella LT2 genome annotation (NCBI accession number AE006468.2 ) or pSLT plasmid annotation (NCBI accession number AE006471.2 ). Moreover, small RNA annotations from Srikumar et al. (2015) were mapped to the LT2 genome. Only sequences that had 100% sequence identity over the full length on LT2 strain were kept. Identical datasets were used for the generation of Figure 8 and Supplementary Figure S6B .
    Figure Legend Snippet: Repartition of enriched RNAs in/on OMVs isolated from the different analyzed in vitro culture conditions. UpSet plot representing the number of enriched RNAs shared between different conditions. One unit represents one expressed gene. The graph was generated using the UpSet R package. Enriched RNAs were determined using DESeq2 , calculating differential presence between intracellular and OMV-associated fractions for each condition. Expressed genes with an average number of reads over 5 for a given set of triplicates, a log 2 Fold Change over 2, and a corresponding adjusted p -value under 0.05 were selected. ncRNA class attribution is defined from the Salmonella LT2 genome annotation (NCBI accession number AE006468.2 ) or pSLT plasmid annotation (NCBI accession number AE006471.2 ). Moreover, small RNA annotations from Srikumar et al. (2015) were mapped to the LT2 genome. Only sequences that had 100% sequence identity over the full length on LT2 strain were kept. Identical datasets were used for the generation of Figure 8 and Supplementary Figure S6B .

    Techniques Used: Isolation, In Vitro, Generated, Plasmid Preparation, Sequencing

    Visualization of intracellular and OMV-related read coverage plots of distinct sRNAs in distinct culture conditions. RNA coverage from sequencing data was visualized with the Integrative Genomics Viewer software (v2.4.8) using default parameters ( Robinson et al., 2011 ). Each plot represents the raw number of reads mapped along the observed sequence, automatically scaled relatively to the highest number of read existing in this portion of the genome. The three plots in dark colors at the top of each window represents the sequencing coverage of OMV-associated fracti ons, in biological triplicate for each condition. On the contrary, the three plots in lighter colors at the bottom of each window show the coverage for the same RNA but from the corresponding intracellular fractions. The arrows under each window precise the genes positions and orientations, according to the data extracted from the Salmonella LT2 genome annotation (NCBI accession number AE006468.2 ) or pSLT plasmid annotation (NCBI accession number AE006471.2 ). The identical coverage patterns observed in both fractions for the displayed genes stand for a native export through OMVs without specific processing or degradation.
    Figure Legend Snippet: Visualization of intracellular and OMV-related read coverage plots of distinct sRNAs in distinct culture conditions. RNA coverage from sequencing data was visualized with the Integrative Genomics Viewer software (v2.4.8) using default parameters ( Robinson et al., 2011 ). Each plot represents the raw number of reads mapped along the observed sequence, automatically scaled relatively to the highest number of read existing in this portion of the genome. The three plots in dark colors at the top of each window represents the sequencing coverage of OMV-associated fracti ons, in biological triplicate for each condition. On the contrary, the three plots in lighter colors at the bottom of each window show the coverage for the same RNA but from the corresponding intracellular fractions. The arrows under each window precise the genes positions and orientations, according to the data extracted from the Salmonella LT2 genome annotation (NCBI accession number AE006468.2 ) or pSLT plasmid annotation (NCBI accession number AE006471.2 ). The identical coverage patterns observed in both fractions for the displayed genes stand for a native export through OMVs without specific processing or degradation.

    Techniques Used: Sequencing, Software, Plasmid Preparation

    RNase protection assay of selected protein-associated sRNAs isolated from OMVs. OMV isolation and enzymatic digestions were performed from SPI-1ind condition as described in Section “Materials and Methods (see section “OMV Purification and RNA Extraction”).” Crude OMV preparation was divided in four fractions. One was kept untouched (Untreated). The second was subjected to the same protocol than the third and fourth fraction without any enzyme added (Control). The third was digested by RNaseA. The fourth was digested by ProteinaseK followed by proteinase inactivation and RNAseA digestion. The protocol was then followed as described in Section “OMV Purification and RNA Extraction” until cDNA libraries from OMV-associated RNAs were obtained End-point PCR was then conducted with the same primers and conditions used previously, for SsrS , CsrC , 10Sa , and rnpB genes. Resulting samples were deposited on a 3% w/v agarose gel in TBE buffer, subjected to electrophoresis and stained with Ethidium Bromide. Most of the signal is still present after enzymatic digestion, showing that the RNAs are protected by the vesicle membrane. –RT controls and positive controls for enzymatic digestions can be seen in Supplementary Figure S5 .
    Figure Legend Snippet: RNase protection assay of selected protein-associated sRNAs isolated from OMVs. OMV isolation and enzymatic digestions were performed from SPI-1ind condition as described in Section “Materials and Methods (see section “OMV Purification and RNA Extraction”).” Crude OMV preparation was divided in four fractions. One was kept untouched (Untreated). The second was subjected to the same protocol than the third and fourth fraction without any enzyme added (Control). The third was digested by RNaseA. The fourth was digested by ProteinaseK followed by proteinase inactivation and RNAseA digestion. The protocol was then followed as described in Section “OMV Purification and RNA Extraction” until cDNA libraries from OMV-associated RNAs were obtained End-point PCR was then conducted with the same primers and conditions used previously, for SsrS , CsrC , 10Sa , and rnpB genes. Resulting samples were deposited on a 3% w/v agarose gel in TBE buffer, subjected to electrophoresis and stained with Ethidium Bromide. Most of the signal is still present after enzymatic digestion, showing that the RNAs are protected by the vesicle membrane. –RT controls and positive controls for enzymatic digestions can be seen in Supplementary Figure S5 .

    Techniques Used: Rnase Protection Assay, Isolation, Purification, RNA Extraction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis, Staining

    Differential abundance of RNAs in OMVs and their intracellular counterparts. (A–E) Scatterplots showing the reads repartition within a given growth condition. Each dot represents the relative abundance of an expressed gene in or attached to the OMVs (vertical axis) and in the intracellular (horizontal axis) fraction for each growth condition used in this study. RNA annotation from the Salmonella LT2 genome (NCBI accession number AE006468.2 ) or pSLT plasmid (NCBI accession number AE006471.2 ) is precized by a color code. Number of reads were normalized by sum normalization for each condition using the decostand function from the vegan R package. Ribosomal RNAs, which were experimentally depleted for intracellular fractions, and removed in silico for OMV fractions, are omitted. (F) Color legend used to underline the RNA class of each annotated transcript in the graphs.
    Figure Legend Snippet: Differential abundance of RNAs in OMVs and their intracellular counterparts. (A–E) Scatterplots showing the reads repartition within a given growth condition. Each dot represents the relative abundance of an expressed gene in or attached to the OMVs (vertical axis) and in the intracellular (horizontal axis) fraction for each growth condition used in this study. RNA annotation from the Salmonella LT2 genome (NCBI accession number AE006468.2 ) or pSLT plasmid (NCBI accession number AE006471.2 ) is precized by a color code. Number of reads were normalized by sum normalization for each condition using the decostand function from the vegan R package. Ribosomal RNAs, which were experimentally depleted for intracellular fractions, and removed in silico for OMV fractions, are omitted. (F) Color legend used to underline the RNA class of each annotated transcript in the graphs.

    Techniques Used: Plasmid Preparation, In Silico

    6) Product Images from "Macrophages attenuate the transcription of CYP1A1 in breast tumor cells and enhance their proliferation"

    Article Title: Macrophages attenuate the transcription of CYP1A1 in breast tumor cells and enhance their proliferation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0209694

    Functional impact of macrophages on breast tumor cells. (A) Enriched biological processes as determined by GO term analysis of the RNA seq data from MΦ-infiltrated and non-infiltrated tumor spheroids. (B) 1 x 10 4 MCF7 cells were seeded in a 96-well plate and incubated with supernatants from MCF7 cells or MΦs. Proliferation was assessed using an IncuCyte S3 system and is presented as relative increase in confluency. Data are presented as means ± SEM (n > 3, * p
    Figure Legend Snippet: Functional impact of macrophages on breast tumor cells. (A) Enriched biological processes as determined by GO term analysis of the RNA seq data from MΦ-infiltrated and non-infiltrated tumor spheroids. (B) 1 x 10 4 MCF7 cells were seeded in a 96-well plate and incubated with supernatants from MCF7 cells or MΦs. Proliferation was assessed using an IncuCyte S3 system and is presented as relative increase in confluency. Data are presented as means ± SEM (n > 3, * p

    Techniques Used: Functional Assay, RNA Sequencing Assay, Incubation

    Tumor cell-specific gene expression changes after macrophage infiltration. (A) Schematic overview of the experimental setup of tumor cell isolation for RNA seq. (B) Purity of tumor cells after removal of CD14 + cells from dissociated tumor spheroids was determined by FACS analysis of tumor cells (EpCAM + ) and immune cells (CD45 + ). Graph is representative of 3 independent experiments. The proportion of immune cells (CD45 + ) was quantified relative to all cells and is given as mean ± SEM (n = 3). (C) Top differentially expressed genes identified by RNA seq analysis of tumor cells from infiltrated relative to non-infiltrated MCF7 tumor spheroids.
    Figure Legend Snippet: Tumor cell-specific gene expression changes after macrophage infiltration. (A) Schematic overview of the experimental setup of tumor cell isolation for RNA seq. (B) Purity of tumor cells after removal of CD14 + cells from dissociated tumor spheroids was determined by FACS analysis of tumor cells (EpCAM + ) and immune cells (CD45 + ). Graph is representative of 3 independent experiments. The proportion of immune cells (CD45 + ) was quantified relative to all cells and is given as mean ± SEM (n = 3). (C) Top differentially expressed genes identified by RNA seq analysis of tumor cells from infiltrated relative to non-infiltrated MCF7 tumor spheroids.

    Techniques Used: Expressing, Cell Isolation, RNA Sequencing Assay, FACS

    7) Product Images from "The long noncoding RNA SPRIGHTLY acts as an intranuclear organizing hub for pre-mRNA molecules"

    Article Title: The long noncoding RNA SPRIGHTLY acts as an intranuclear organizing hub for pre-mRNA molecules

    Journal: Science Advances

    doi: 10.1126/sciadv.1602505

    SPRIGHTLY RNA secondary structure and its binding partners. ( A ) The secondary structure of SPRIGHTLY was determined by RNA structure with the constraints of SHAPE-seq reactivity data. Nucleotides are colored by their normalized reactivities ρ. The probe sequences are labeled P1 to P12. In the text, the 12 primers are termed in three sets: Set D1 is represented by probes P1 to P4, D2 is represented by probes P5 to P8, and D3 is represented by probes P9 to P12. The core pseudoknotted domain overlaps D1, D2, and D3. ( B ) Histogram of normalized SHAPE-seq reactivities as a function of nucleotide position of SPRIGHTLY . ( C ) The distribution of RNA sequences within gene bodies corresponding to dChIRP MACS peaks pulled down by the three sets of SPRIGHTLY probes, D1, D2, and D3. dChIRP MACS peaks found in the exonic region including promoter-TSS, exon, 3′UTR, and TTS were plotted. The aggregate plots of RNA dChIRP sequences peaks show the enriched regions distributed across 5000 base pairs (bp) upstream of gene bodies and 5000 bp downstream of the genes. The shades represent the SEM. Green peaks represent RNA pulled down by probes of D1, orange peaks represent RNA pulled down by probes of D2, and purple peaks represent RNA pulled down by probes of D3. ( D ) SPRIGHTLY binding partner RNAs determined by common MACS peaks. The MACS peaks were mapped to their corresponding genomic loci, and the number of genes was counted. If MACS peaks from individual dChIRP sequencing overlapped or mapped to same gene, then those genes were regarded as SPRIGHTLY binding partners. Six genes have MACS peaks common to all three regions, suggesting that those six genes are most likely to interact with SPRIGHTLY . ( E ) SPRIGHTLY dChIRP specifically enriches the intronic regions of six genes. SPRIGHTLY dChIRP samples were analyzed by qPCR using primers for representative MACS peak of each gene or using primers for exon-exon junctions. Each intronic region corresponding to MACS peak was enriched > 5- to 800-fold over the abundant glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) mRNA. An average of three technical replicates ± SD is shown. ( F ) The integrated network of six RNA molecules that bind to SPRIGHTLY was constructed by querying integrated gene interaction network data. Green interaction edges represent high-confidence genetic interaction data from Lin et al . ( 51 ), and black dashed edges represent consensus miRNA target sequences ( 52 ).
    Figure Legend Snippet: SPRIGHTLY RNA secondary structure and its binding partners. ( A ) The secondary structure of SPRIGHTLY was determined by RNA structure with the constraints of SHAPE-seq reactivity data. Nucleotides are colored by their normalized reactivities ρ. The probe sequences are labeled P1 to P12. In the text, the 12 primers are termed in three sets: Set D1 is represented by probes P1 to P4, D2 is represented by probes P5 to P8, and D3 is represented by probes P9 to P12. The core pseudoknotted domain overlaps D1, D2, and D3. ( B ) Histogram of normalized SHAPE-seq reactivities as a function of nucleotide position of SPRIGHTLY . ( C ) The distribution of RNA sequences within gene bodies corresponding to dChIRP MACS peaks pulled down by the three sets of SPRIGHTLY probes, D1, D2, and D3. dChIRP MACS peaks found in the exonic region including promoter-TSS, exon, 3′UTR, and TTS were plotted. The aggregate plots of RNA dChIRP sequences peaks show the enriched regions distributed across 5000 base pairs (bp) upstream of gene bodies and 5000 bp downstream of the genes. The shades represent the SEM. Green peaks represent RNA pulled down by probes of D1, orange peaks represent RNA pulled down by probes of D2, and purple peaks represent RNA pulled down by probes of D3. ( D ) SPRIGHTLY binding partner RNAs determined by common MACS peaks. The MACS peaks were mapped to their corresponding genomic loci, and the number of genes was counted. If MACS peaks from individual dChIRP sequencing overlapped or mapped to same gene, then those genes were regarded as SPRIGHTLY binding partners. Six genes have MACS peaks common to all three regions, suggesting that those six genes are most likely to interact with SPRIGHTLY . ( E ) SPRIGHTLY dChIRP specifically enriches the intronic regions of six genes. SPRIGHTLY dChIRP samples were analyzed by qPCR using primers for representative MACS peak of each gene or using primers for exon-exon junctions. Each intronic region corresponding to MACS peak was enriched > 5- to 800-fold over the abundant glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) mRNA. An average of three technical replicates ± SD is shown. ( F ) The integrated network of six RNA molecules that bind to SPRIGHTLY was constructed by querying integrated gene interaction network data. Green interaction edges represent high-confidence genetic interaction data from Lin et al . ( 51 ), and black dashed edges represent consensus miRNA target sequences ( 52 ).

    Techniques Used: Binding Assay, Labeling, Magnetic Cell Separation, Sequencing, Real-time Polymerase Chain Reaction, Construct

    8) Product Images from "Determinants of tRNA Recognition by the Radical SAM Enzyme RlmN"

    Article Title: Determinants of tRNA Recognition by the Radical SAM Enzyme RlmN

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0167298

    Interrogation of chimeric tRNA substrates. (A) A representative 2D structure of tRNA. (B) In silico predicted representation of chimeric tRNA used in this study. Elements derived from substrate tRNA Gln UUG are in blue, while those deriving from non-substrate tRNA Gly CCC are in red. (C) End-point methylation of chimeric tRNA and tRNA Gln UUG ACSL RNA. Bars represent the mean ± s.d. of at least two replicates.
    Figure Legend Snippet: Interrogation of chimeric tRNA substrates. (A) A representative 2D structure of tRNA. (B) In silico predicted representation of chimeric tRNA used in this study. Elements derived from substrate tRNA Gln UUG are in blue, while those deriving from non-substrate tRNA Gly CCC are in red. (C) End-point methylation of chimeric tRNA and tRNA Gln UUG ACSL RNA. Bars represent the mean ± s.d. of at least two replicates.

    Techniques Used: In Silico, Derivative Assay, Countercurrent Chromatography, Methylation

    RlmN mediated methylation of in vitro transcribed tRNAs. (A) End-point methylation of in vitro transcribed RNAs. Bars represent the mean of at least two replicates ± s.d. (B) Radio HPLC analysis of nucleosides obtained by methylation and total RNA digestion of select tRNA substrates. Radioactive signal co-elutes with an authentic m 2 A standard. (C) Evaluation of reaction requirements for methylation of in vitro transcribed tRNA Gln UUG . Bars represent the mean of two replicates ± s.d.
    Figure Legend Snippet: RlmN mediated methylation of in vitro transcribed tRNAs. (A) End-point methylation of in vitro transcribed RNAs. Bars represent the mean of at least two replicates ± s.d. (B) Radio HPLC analysis of nucleosides obtained by methylation and total RNA digestion of select tRNA substrates. Radioactive signal co-elutes with an authentic m 2 A standard. (C) Evaluation of reaction requirements for methylation of in vitro transcribed tRNA Gln UUG . Bars represent the mean of two replicates ± s.d.

    Techniques Used: Methylation, In Vitro, High Performance Liquid Chromatography

    9) Product Images from "Osterix functions downstream of anti-Müllerian hormone signaling to regulate Müllerian duct regression"

    Article Title: Osterix functions downstream of anti-Müllerian hormone signaling to regulate Müllerian duct regression

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1721793115

    A transcriptome screen for male-specific genes expressed in Amhr2 -expressing Müllerian duct (MD) mesenchyme. ( A ) Breeding scheme to isolate Amhr2 -marked MD mesenchyme cells from E14.5 male and female embryos. The Amhr2-Cre allele is expressed in MD mesenchyme cells and somatic cells of the gonad. Mesonephroi containing the MD (arrowheads) and WDs were isolated by removing the gonads. Subsequently, YFP+ MD mesenchyme cells were isolated by FACS. ( B ) Representative plot forward scatter versus YFP emission (530/30-nm bandpass filter) from five male Amhr2 Cre/+ ; R26R YFP/+ mesenophros pairs to isolate YFP+ cells. ( C ) Representative confocal image of sorted cell fractions. ( D ) High-sensitivity DNA chip analysis of index pooled cDNA libraries for RNA-seq. O, ovary; T, testis. (Magnification: A , 40×; C , 200×.)
    Figure Legend Snippet: A transcriptome screen for male-specific genes expressed in Amhr2 -expressing Müllerian duct (MD) mesenchyme. ( A ) Breeding scheme to isolate Amhr2 -marked MD mesenchyme cells from E14.5 male and female embryos. The Amhr2-Cre allele is expressed in MD mesenchyme cells and somatic cells of the gonad. Mesonephroi containing the MD (arrowheads) and WDs were isolated by removing the gonads. Subsequently, YFP+ MD mesenchyme cells were isolated by FACS. ( B ) Representative plot forward scatter versus YFP emission (530/30-nm bandpass filter) from five male Amhr2 Cre/+ ; R26R YFP/+ mesenophros pairs to isolate YFP+ cells. ( C ) Representative confocal image of sorted cell fractions. ( D ) High-sensitivity DNA chip analysis of index pooled cDNA libraries for RNA-seq. O, ovary; T, testis. (Magnification: A , 40×; C , 200×.)

    Techniques Used: Expressing, Isolation, FACS, Chromatin Immunoprecipitation, RNA Sequencing Assay

    10) Product Images from "Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses"

    Article Title: Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2016.01588

    Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including DNase digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.
    Figure Legend Snippet: Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including DNase digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.

    Techniques Used: Environmental Sampling, Purification, Real-time Polymerase Chain Reaction, Lysis

    Removal of gDNA by consecutive DNase digestions of total nucleic acids (TNA) extracted from 45 Å soil samples. The soil had been exposed to different oxygen regimes (here called Treatments 1, 2, and 3), for details see section “Materials and Methods.” The soils were incubated anoxically to stimulate denitrification gene expression, and samples were taken at time int ervals. TNA was extracted using the optimized and simplified method, and the nosZ was quantified by qPCR. (A) After extraction via the optimized method, all samples were tested for the presence of DNA. Neither the different oxygen regimes nor the stimulation of gene expression affected the number of nosZ genes in the gDNA from the different samples. (B) The first digest removed most amplifiable genomic DNA (gDNA) present. (C) The second DNase treatment removed amplifiable gDNA in all samples. There was no relationship between the starting DNA quantity and the success of complete gDNA removal ( R 2 = 0.0189). This highlights the importance of checking all RNA samples and not only representative samples, as there may be high variability among samples from the same source and extraction procedure.
    Figure Legend Snippet: Removal of gDNA by consecutive DNase digestions of total nucleic acids (TNA) extracted from 45 Å soil samples. The soil had been exposed to different oxygen regimes (here called Treatments 1, 2, and 3), for details see section “Materials and Methods.” The soils were incubated anoxically to stimulate denitrification gene expression, and samples were taken at time int ervals. TNA was extracted using the optimized and simplified method, and the nosZ was quantified by qPCR. (A) After extraction via the optimized method, all samples were tested for the presence of DNA. Neither the different oxygen regimes nor the stimulation of gene expression affected the number of nosZ genes in the gDNA from the different samples. (B) The first digest removed most amplifiable genomic DNA (gDNA) present. (C) The second DNase treatment removed amplifiable gDNA in all samples. There was no relationship between the starting DNA quantity and the success of complete gDNA removal ( R 2 = 0.0189). This highlights the importance of checking all RNA samples and not only representative samples, as there may be high variability among samples from the same source and extraction procedure.

    Techniques Used: Incubation, Expressing, Real-time Polymerase Chain Reaction

    11) Product Images from "COMRADES determines in vivo RNA structures and interactions"

    Article Title: COMRADES determines in vivo RNA structures and interactions

    Journal: Nature methods

    doi: 10.1038/s41592-018-0121-0

    Host-virus RNA-RNA interactions. a, Human RNA species interacting with the ZIKV genome. Mean and s.d. of 3 independent experiments are shown. b, Probed interactions between the ZIKV genome and specific miRNAs in COMRADES and control samples. c, COMRADES determined base-pairing between ZIKV and miR-21. d, A model of the ZIKV 5' CS engaged in three separate functions. Ribosome and nascent polypeptide are marked in green.
    Figure Legend Snippet: Host-virus RNA-RNA interactions. a, Human RNA species interacting with the ZIKV genome. Mean and s.d. of 3 independent experiments are shown. b, Probed interactions between the ZIKV genome and specific miRNAs in COMRADES and control samples. c, COMRADES determined base-pairing between ZIKV and miR-21. d, A model of the ZIKV 5' CS engaged in three separate functions. Ribosome and nascent polypeptide are marked in green.

    Techniques Used:

    12) Product Images from "Macrophages attenuate the transcription of CYP1A1 in breast tumor cells and enhance their proliferation"

    Article Title: Macrophages attenuate the transcription of CYP1A1 in breast tumor cells and enhance their proliferation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0209694

    Tumor cell-specific gene expression changes after macrophage infiltration. (A) Schematic overview of the experimental setup of tumor cell isolation for RNA seq. (B) Purity of tumor cells after removal of CD14+ cells from dissociated tumor spheroids was determined by FACS analysis of tumor cells (EpCAM+ ) and immune cells (CD45+ ). Graph is representative of 3 independent experiments. The proportion of immune cells (CD45+ ) was quantified relative to all cells and is given as mean ± SEM (n = 3). (C) Top differentially expressed genes identified by RNA seq analysis of tumor cells from infiltrated relative to non-infiltrated MCF7 tumor spheroids.
    Figure Legend Snippet: Tumor cell-specific gene expression changes after macrophage infiltration. (A) Schematic overview of the experimental setup of tumor cell isolation for RNA seq. (B) Purity of tumor cells after removal of CD14+ cells from dissociated tumor spheroids was determined by FACS analysis of tumor cells (EpCAM+ ) and immune cells (CD45+ ). Graph is representative of 3 independent experiments. The proportion of immune cells (CD45+ ) was quantified relative to all cells and is given as mean ± SEM (n = 3). (C) Top differentially expressed genes identified by RNA seq analysis of tumor cells from infiltrated relative to non-infiltrated MCF7 tumor spheroids.

    Techniques Used: Expressing, Cell Isolation, RNA Sequencing Assay, FACS

    13) Product Images from "The Effect of DNA Topology on Observed Rates of R-Loop Formation and DNA Strand Cleavage by CRISPR Cas12a"

    Article Title: The Effect of DNA Topology on Observed Rates of R-Loop Formation and DNA Strand Cleavage by CRISPR Cas12a

    Journal: Genes

    doi: 10.3390/genes10020169

    Measurement of R-loop formation by LbCas12a using a magnetic tweezers assay. ( A ) DNA protospacer sequence (black) and CRISPR RNA (crRNA) showing the G-residues from in vitro transcription (brown), the pseudoknot (blue) and spacer (red). ( B ) Principle of the MT assay. See main text. ( C ) R-loop cycling experiment (1 turn s −1 ) in the presence of 5 nM Cas12a:crRNA. Raw DNA length taken at 60 Hz (grey). Data smoothed by a 1 Hz moving average (dark colors). DNA is negatively supercoiled at 0.3 pN (red) to induce R-loop formation (in), followed by positive supercoiling to probe R-loop formation (blue), resulting in R-loop dissociation (out). Rot 0 are points where DNA turns are zero. ( D ) Overlay of R-loop cycles ( N = 22) for negative supercoiling (in events) and positive supercoiling (out events). Cycles without Cas12a are in grey. Data was smoothed by a 1 Hz moving average. ( i ) and ( ii ) show rotation curve shifts due to captured R-loops. ( E ) Rotation curve shift due to R-loop events ( i ). Average = 1.87 ± 0.27 turns (errors = SD). ( F ) Examples of repetitive R-loop formation cycling (at 10 turns s −1 ) to measure R-loop formation times. Raw and 1 Hz smoothed data are shown. ( G ) Mean R-loop formation/dissociation times and standard error ( N = 40 to 52) as a function of torque [ 29 ]. Solid lines are fits to Equation (1) ( Table 1 ) [ 27 ]. ( H ) Inverted cumulative probability over time for R-loop formation (left) and dissociation (right) used to calculate mean times in panel F.
    Figure Legend Snippet: Measurement of R-loop formation by LbCas12a using a magnetic tweezers assay. ( A ) DNA protospacer sequence (black) and CRISPR RNA (crRNA) showing the G-residues from in vitro transcription (brown), the pseudoknot (blue) and spacer (red). ( B ) Principle of the MT assay. See main text. ( C ) R-loop cycling experiment (1 turn s −1 ) in the presence of 5 nM Cas12a:crRNA. Raw DNA length taken at 60 Hz (grey). Data smoothed by a 1 Hz moving average (dark colors). DNA is negatively supercoiled at 0.3 pN (red) to induce R-loop formation (in), followed by positive supercoiling to probe R-loop formation (blue), resulting in R-loop dissociation (out). Rot 0 are points where DNA turns are zero. ( D ) Overlay of R-loop cycles ( N = 22) for negative supercoiling (in events) and positive supercoiling (out events). Cycles without Cas12a are in grey. Data was smoothed by a 1 Hz moving average. ( i ) and ( ii ) show rotation curve shifts due to captured R-loops. ( E ) Rotation curve shift due to R-loop events ( i ). Average = 1.87 ± 0.27 turns (errors = SD). ( F ) Examples of repetitive R-loop formation cycling (at 10 turns s −1 ) to measure R-loop formation times. Raw and 1 Hz smoothed data are shown. ( G ) Mean R-loop formation/dissociation times and standard error ( N = 40 to 52) as a function of torque [ 29 ]. Solid lines are fits to Equation (1) ( Table 1 ) [ 27 ]. ( H ) Inverted cumulative probability over time for R-loop formation (left) and dissociation (right) used to calculate mean times in panel F.

    Techniques Used: Sequencing, CRISPR, In Vitro

    14) Product Images from "High expression of CAI2, a 9p21-embedded long non-coding RNA, contributes to advanced stage neuroblastoma"

    Article Title: High expression of CAI2, a 9p21-embedded long non-coding RNA, contributes to advanced stage neuroblastoma

    Journal:

    doi: 10.1158/0008-5472.CAN-13-3447

    The discovery of CAI2 as a tumor expressed ncRNA (A) The 5’ and 3’ RACE smear (RS) and RACE band (RB) cut out of an agarose gel and cloned are boxed. A 100bp molecular weight ladder is included in the 3’ RACE figure; (B) The fragment of 9p21 containing the genomic orientation of ARF, p16, p16γ and CAI2 exons and transcripts are illustrated, with the RACE primers and results illustrated. ARF exon 1 (X1β), p16 exon 1 (X1α), p16/ARF exon 2 (X2) and 3 coding (X3) and exon γ (Xγ) are shown; (C) The ORFs greater than 40 AA are illustrated relative to the entire CAI2 transcript. The many ORFs smaller than 40 AA are unlabeled and shaded in grey; (D) CAI2 , ORF2 and p16 were assessed for protein production using the Promega TNT Coupled Reticulocyte Lysate Systems. The luciferase control yielded a sharp band of the correct molecular weight (lane 1). p16 yielded a faint band at the correct molecular weight of 16 kDa (lane 2), consistent with the paucity of methionine (N=5) and cysteine (N=1) residues. Neither ORF2 (lane 3) nor CAI2 in either the sense (lane 4) or antisense (lane 5) orientations yielded any evidence of a protein; (E) CAI2 was detected only in reverse-transcribed RNA. And though CAI2 sequence could be amplified from genomic DNA, amplification was fully abolished by the same DNAse treatment the RNA samples were subjected to; (F) Representative non-quantitative amplifications demonstrate robust expression of CAI2 in most tumor cell lines (HL60, NB20 and HeLa), though some tumor cell lines (PCL1643) and most normal samples (MNC) express CAI2 only weakly; Molt4 are deleted (summarized in ).
    Figure Legend Snippet: The discovery of CAI2 as a tumor expressed ncRNA (A) The 5’ and 3’ RACE smear (RS) and RACE band (RB) cut out of an agarose gel and cloned are boxed. A 100bp molecular weight ladder is included in the 3’ RACE figure; (B) The fragment of 9p21 containing the genomic orientation of ARF, p16, p16γ and CAI2 exons and transcripts are illustrated, with the RACE primers and results illustrated. ARF exon 1 (X1β), p16 exon 1 (X1α), p16/ARF exon 2 (X2) and 3 coding (X3) and exon γ (Xγ) are shown; (C) The ORFs greater than 40 AA are illustrated relative to the entire CAI2 transcript. The many ORFs smaller than 40 AA are unlabeled and shaded in grey; (D) CAI2 , ORF2 and p16 were assessed for protein production using the Promega TNT Coupled Reticulocyte Lysate Systems. The luciferase control yielded a sharp band of the correct molecular weight (lane 1). p16 yielded a faint band at the correct molecular weight of 16 kDa (lane 2), consistent with the paucity of methionine (N=5) and cysteine (N=1) residues. Neither ORF2 (lane 3) nor CAI2 in either the sense (lane 4) or antisense (lane 5) orientations yielded any evidence of a protein; (E) CAI2 was detected only in reverse-transcribed RNA. And though CAI2 sequence could be amplified from genomic DNA, amplification was fully abolished by the same DNAse treatment the RNA samples were subjected to; (F) Representative non-quantitative amplifications demonstrate robust expression of CAI2 in most tumor cell lines (HL60, NB20 and HeLa), though some tumor cell lines (PCL1643) and most normal samples (MNC) express CAI2 only weakly; Molt4 are deleted (summarized in ).

    Techniques Used: Agarose Gel Electrophoresis, Clone Assay, Molecular Weight, Luciferase, Sequencing, Amplification, Expressing

    15) Product Images from "A Novel Strategy for Exploitation of Host RNase E Activity by a Marine Cyanophage"

    Article Title: A Novel Strategy for Exploitation of Host RNase E Activity by a Marine Cyanophage

    Journal:

    doi: 10.1534/genetics.115.183475

    Regulation of Prochlorococcus MED4 RNase E during the course of P-SSP7 infection. (A) cDNA reads from total RNA (−TEX) or RNA enriched in primary transcripts of noninfected (+TEX) or infected cells (+TEX-INF) mapped to the rne locus encoding RNase
    Figure Legend Snippet: Regulation of Prochlorococcus MED4 RNase E during the course of P-SSP7 infection. (A) cDNA reads from total RNA (−TEX) or RNA enriched in primary transcripts of noninfected (+TEX) or infected cells (+TEX-INF) mapped to the rne locus encoding RNase

    Techniques Used: Infection

    16) Product Images from "Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair"

    Article Title: Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair

    Journal: eLife

    doi: 10.7554/eLife.33761

    Covalent linkage of the DNA repair template to the Cas9 RNP complex. ( a ) Quantification of HDR rates with DNA repair templates of different lengths by FACS. ( b ) Quantification of sgRNA Sp Cas9 (mutRFP) editing efficiency in the reporter cell line by FACS. ( c ) SYBR-Gold stained denaturing PAGE gel. Proportion of shifted 81-mer amino-modified oligos after the coupling reaction with different concentrations of BG-GLA-NHS building blocks. ( d ) Negative ion ESI mass spectra of HPLC purified BG-coupled (top panel) and uncoupled (bottom panel) oligos shown in Figure 3b . ( e,f ) SDS-PAGE gels of Sp Cas9-SNAP ( e ) and the Sa dCas9-SNAP ( f ) proteins labeled with BG-Vista Green (upper panels). Subsequently gels were silver stained for protein detection (lower panels). ( g,h ) Denaturing RNA gels (2% MOPS) with in vitro transcribed sgRNAs. ( g ) sgRNA used to create the DSB in the mutRFP locus (sgRNA Sp Cas9 (mutRFP)). ( h ) sgRNAs used to bind catalytically inactive Sa dCas9 to regions upstream (sgRNA Sa dCas9 (mutRFP)−1 and sgRNA Sa dCas9 (mutRFP)−2) and downstream (sgRNA Sa dCas9 (mutRFP)−3 and sgRNA Sa dCas9 (mutRFP)−4) of the mutRFP locus. Box and whiskers plot: min to max (**p
    Figure Legend Snippet: Covalent linkage of the DNA repair template to the Cas9 RNP complex. ( a ) Quantification of HDR rates with DNA repair templates of different lengths by FACS. ( b ) Quantification of sgRNA Sp Cas9 (mutRFP) editing efficiency in the reporter cell line by FACS. ( c ) SYBR-Gold stained denaturing PAGE gel. Proportion of shifted 81-mer amino-modified oligos after the coupling reaction with different concentrations of BG-GLA-NHS building blocks. ( d ) Negative ion ESI mass spectra of HPLC purified BG-coupled (top panel) and uncoupled (bottom panel) oligos shown in Figure 3b . ( e,f ) SDS-PAGE gels of Sp Cas9-SNAP ( e ) and the Sa dCas9-SNAP ( f ) proteins labeled with BG-Vista Green (upper panels). Subsequently gels were silver stained for protein detection (lower panels). ( g,h ) Denaturing RNA gels (2% MOPS) with in vitro transcribed sgRNAs. ( g ) sgRNA used to create the DSB in the mutRFP locus (sgRNA Sp Cas9 (mutRFP)). ( h ) sgRNAs used to bind catalytically inactive Sa dCas9 to regions upstream (sgRNA Sa dCas9 (mutRFP)−1 and sgRNA Sa dCas9 (mutRFP)−2) and downstream (sgRNA Sa dCas9 (mutRFP)−3 and sgRNA Sa dCas9 (mutRFP)−4) of the mutRFP locus. Box and whiskers plot: min to max (**p

    Techniques Used: FACS, Staining, Polyacrylamide Gel Electrophoresis, Modification, High Performance Liquid Chromatography, Purification, SDS Page, Labeling, In Vitro

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    Incubation:

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    Infection:

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    Magnetic Beads:

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    Polymerase Chain Reaction:

    Article Title: Staphylococcus aureus Cas9 is a multiple-turnover enzyme
    Article Snippet: S. pyogenes Cas9 (# M0386M), EnGen sgRNA Synthesis Kit, S. pyogenes (# E3322S), HiScribe T7 High Yield RNA Synthesis Kit (E2040S), 2× RNA Loading Dye (# B0363S), Nucleoside Digestion Mix (M0649S), Q5 Hot Start High-Fidelity 2× Master Mix (# M0494L), Monarch PCR & DNA Cleanup Kit (# T1030L), Proteinase K (# P8107S), Shrimp Alkaline Phosphatase (# M0371S), T4 Polynucleotide Kinase (# M0201S), Streptavidin Magnetic Beads (# S1420S), and NEBuffer 3.1 (# B7203S) with a 1× composition of 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , 100 μg/ml BSA, pH 7.9 at 25°C were all from New England Biolabs. .. The Zymo RNA Clean & Concentrator-5 kit (#R1016) was purchased from Zymo Research.

    Article Title: Impact of CodY protein on metabolism, sporulation and virulence in Clostridioides difficile ribotype 027
    Article Snippet: Removal of DNA was considered successful if no PCR product was detected after 30 cycles of amplification using oND52/53 primers specific for genes encoding 16s rRNA. .. Fragmented RNA was purified and concentrated using the RNA Clean & Concentrator-5 kit (Zymo Research Corporation).

    Article Title: mRNA processing in mutant zebrafish lines generated by chemical and CRISPR-mediated mutagenesis produces unexpected transcripts that escape nonsense-mediated decay
    Article Snippet: To genotype individual samples, residual gDNA in the unpurified RNA samples was PCR amplified and sent for Sanger sequencing. .. After genotypes were determined (SnapGene Viewer to view peak trace files), RNA samples were DNAase I-treated and purified (RNA Clean and Concentrator, Zymo Research), served as templates for cDNA synthesis (iScript cDNA Synthesis Kit, Bio-Rad), and ultimately used in qPCR studies to analyze transcript levels.

    Article Title: Endothelial Cell Stimulation by Candida albicans
    Article Snippet: Add 2 units of DNase I to 5 μg of each RNA sample and incubate at 37°C for 30 min. Clean up the RNA samples with RNA clean-up Kit (such as kit R1015, from Zymo Research, Inc., Orange, CA). .. Add 2 units of DNase I to 5 μg of each RNA sample and incubate at 37°C for 30 min. Clean up the RNA samples with RNA clean-up Kit (such as kit R1015, from Zymo Research, Inc., Orange, CA).

    Article Title: RNA-guided assembly of Rev-RRE nuclear export complexes
    Article Snippet: RNA samples from all constructs were prepared by in vitro transcription using T7 polymerase, with either linearized plasmid or PCR product DNA as templates. .. For SHAPE analysis RNA samples were column-purified using RNA Clean & Concentrator-25 (Zymo Research, Irvine, CA, USA), and for SAXS experiments RNA samples were gel-purified and washed multiple times through filtration.

    Article Title: Diverse electron sources support denitrification under hypoxia in the obligate methanotroph Methylomicrobium album strain BG8
    Article Snippet: The total RNA was then column-purified using RNA clean & concentrator (Zymo Research). .. Residual genomic DNA contamination was assessed by quantitative PCR (qPCR) targeting norB1 or nirS genes (primers listed in Table ).

    Purification:

    Article Title: A Synonymous Mutation Upstream of the Gene Encoding a Weak-Link Enzyme Causes an Ultrasensitive Response in Growth Rate
    Article Snippet: Frozen pellets were thawed, and the RNA was purified using RNeasy spin columns (Qiagen) according to the manufacturer's protocol. .. An additional DNase treatment performed using Turbo DNase (Life Technologies) according to the manufacturer's protocol was followed by purification with RNA Clean & Concentrator-5 columns (Zymo Research). .. RNA concentration was determined using the Qubit RNA HS assay kit with a Qubit 3.0 fluorometer (Life Technologies).

    Article Title: The YTH Domain Protein ECT2 Is an m6A Reader Required for Normal Trichome Branching in Arabidopsis
    Article Snippet: RNAs were precipitated by centrifugation, resuspended in 350 μL of RTL buffer (Qiagen RNEasy Micro kit), purified according to the manufacturer’s instructions, and eluted in 15 μL of RNase-free water. .. The RNA solution was concentrated into a 6 μL volume of RNase-free water using the RNA clean and concentrator kit R1015 from Zymo Research.

    Article Title: YTHDF3 facilitates translation and decay of N6-methyladenosine-modified RNA
    Article Snippet: Sixty million HeLa cells stably overexpressing Flag-HA-tagged YTHDF3 were subjected to RIP procedure. .. Input, flow-through, and YTHDF3-bound RNA were purified with Trizol reagent. mRNAs of the three portions were further purified by depleting rRNA with RiboMinus Eukaryote Kit v2 (Ambion) followed by depleting tRNA with RNA Clean and Concentrator-5 (Zymo Research, 200 nt cutoff protocol). .. About 50 ng purified mRNA of each sample were subjected to LC-MS/MS quantification of m6 A levels as described above , .

    Article Title: Staphylococcus aureus Cas9 is a multiple-turnover enzyme
    Article Snippet: Both S. pyogenes and S. aureus Cas9 were purified at New England Biolabs using standard liquid chromatography protein purification techniques. .. The Zymo RNA Clean & Concentrator-5 kit (#R1016) was purchased from Zymo Research.

    Article Title: Impact of CodY protein on metabolism, sporulation and virulence in Clostridioides difficile ribotype 027
    Article Snippet: Only RNA samples with an RNA integrity number > 8 were used for library construction. rRNA was depleted from DNA-free RNA preparations using the RiboZero Magnetic kit (Gram-positive kit; Epicentre). mRNAs were fragmented using the NEB Next RNA Fragmentation Module (New England Biolabs) and further assessed using the Bioanalyzer and RNA Pico Chip. .. Fragmented RNA was purified and concentrated using the RNA Clean & Concentrator-5 kit (Zymo Research Corporation). .. First-strand cDNA was synthesized using SuperScript III reverse transcriptase (Life Technologies, Inc.); actinomycin D (8 μg) was included in each reaction to prevent spurious second-strand synthesis.

    Article Title: Metabolomic and transcriptomic changes underlying cold and anaerobic stresses after storage of table grapes
    Article Snippet: The pellet was then placed in 30 µl of DEPC water and mixed by pipetting. .. The RNA was purified with an RNA Clean and Concentrator Kit (Zymo Research, Irvine, CA, USA). .. RNA was sequenced by the Technion Genome Center (Haifa, Israel) following integrity analysis with the TapeStation (Agilent), and sequencing libraries were generated with the TrueSeq RNA Sample Prep Kit v2 (Illumina) according to the manufacturer’s protocol.

    Article Title: H2A.Z controls the stability and mobility of nucleosomes to regulate expression of the LH genes
    Article Snippet: The reactions were quenched by addition of 10 mM EDTA. .. RNA was purified using RNA clean and concentrator-5 kit (Zymo research) with subsequent on-column DNase I (Zymo research) treatment according to the manufacturer's instructions. .. After purification, RNase inhibitor (New England Biolabs) was added and samples were treated again with 1 U of DNase I (Invitrogen) to eliminate DNA template residuals.

    Article Title: mRNA processing in mutant zebrafish lines generated by chemical and CRISPR-mediated mutagenesis produces unexpected transcripts that escape nonsense-mediated decay
    Article Snippet: To genotype individual samples, residual gDNA in the unpurified RNA samples was PCR amplified and sent for Sanger sequencing. .. After genotypes were determined (SnapGene Viewer to view peak trace files), RNA samples were DNAase I-treated and purified (RNA Clean and Concentrator, Zymo Research), served as templates for cDNA synthesis (iScript cDNA Synthesis Kit, Bio-Rad), and ultimately used in qPCR studies to analyze transcript levels. .. qPCR methods included SYBR Green-based methods (Sigma-Aldrich, abca1a , creb3l3a , smyd1a ) and Taqman gene expression assays (ThermoFisher Scientific; cd36 , slc27a2a , and abca1b ). ef1α (for smyd1a ) or 18s rRNA (for all others) levels were used as reference genes.

    Article Title: Endothelial Cell Stimulation by Candida albicans
    Article Snippet: Add 2 units of DNase I to 5 μg of each RNA sample and incubate at 37°C for 30 min. Clean up the RNA samples with RNA clean-up Kit (such as kit R1015, from Zymo Research, Inc., Orange, CA). .. Add 2 units of DNase I to 5 μg of each RNA sample and incubate at 37°C for 30 min. Clean up the RNA samples with RNA clean-up Kit (such as kit R1015, from Zymo Research, Inc., Orange, CA).

    Article Title: Telomeric Repeat-Containing RNAs (TERRA) Decrease in Squamous Cell Carcinoma of the Head and Neck Is Associated with Worsened Clinical Outcome
    Article Snippet: RNA samples were treated twice with 1 U DNase I (Promega, Madison, WI, USA) per μg RNA at 37 °C for 30 min. .. Following each DNase treatment after, the RNA was purified with the RNA Clean and Concentration kit (Zymo Research, Irvine, CA, USA) according to manufacturer’s instruction. .. All samples were diluted in three volumes of denaturation solution (26% formaldehyde, 7% of 20× SSC and 24% formamide) and incubated at 65 °C for 5 min, then, one volume of 20× SSC was added.

    Article Title: RNA-guided assembly of Rev-RRE nuclear export complexes
    Article Snippet: For SHAPE analysis RNA samples were column-purified using RNA Clean & Concentrator-25 (Zymo Research, Irvine, CA, USA), and for SAXS experiments RNA samples were gel-purified and washed multiple times through filtration. .. For SHAPE-Seq experiments, barcodes on the RRE molecules were introduced by PCR and placed within the 3′ SHAPE handle as previously described ( ; ).

    Article Title: Diverse electron sources support denitrification under hypoxia in the obligate methanotroph Methylomicrobium album strain BG8
    Article Snippet: Total nucleic acid was purified according to manufacturer’s instructions with the following modifications: 6 U proteinase K (Qiagen) were added to the cell lysis step and the total precipitated nucleic acid was treated with 30 units of DNase I (Ambion). .. The total RNA was then column-purified using RNA clean & concentrator (Zymo Research).

    Article Title: Histone deacetylase 3 associates with MeCP2 to regulate FOXO and social behavior
    Article Snippet: Tissue was rapidly frozen using liquid nitrogen and stored at −80 °C, and RNA extracted using Trizol according to manufacturers protocol (Invitrogen). .. RNA (3 μg) was DNase I treated (4 U, Worthington Biochemical Corporation), purified using RNA Clean and Concentrator-5 Kit (Zymo Research) according to manufacturers’ instructions and eluted with 14 μl DEPC-treated water. .. For each sample, 1 μg RNA was reverse transcribed in a 20 μl reaction volume containing random hexamer mix and Superscript III reverse transcriptase (50 U, Invitrogen) at 50 °C for 1 h. First strand cDNAs were diluted 1:10 and 1 μl were used for qRT-PCR amplification in a 20 μl reaction (SsoFast EvaGreen Supermix, Bio-Rad) containing primers (0.2 μM).

    Article Title: Vaccine Protection Against Zika Virus from Brazil
    Article Snippet: The wildtype ZIKV BeH815744 Cap gene was utilized as a standard and was cloned into pcDNA3.1+, and the AmpliCap-Max T7 High Yield Message Maker Kit was used to transcribe RNA (Cellscript, WI, USA). .. RNA was purified using the RNA clean and concentrator kit (Zymo Research, CA, USA), and RNA quality and concentration was assessed by the BIDMC Molecular Core Facility. .. Log dilutions of the RNA standard were reverse transcribed and included with each RT-PCR assay.

    Dot Blot:

    Article Title: The YTH Domain Protein ECT2 Is an m6A Reader Required for Normal Trichome Branching in Arabidopsis
    Article Snippet: Paragraph title: RIP and m6 A Dot Blot Assays ... The RNA solution was concentrated into a 6 μL volume of RNase-free water using the RNA clean and concentrator kit R1015 from Zymo Research.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Vaccine Protection Against Zika Virus from Brazil
    Article Snippet: Paragraph title: RT-PCR ... RNA was purified using the RNA clean and concentrator kit (Zymo Research, CA, USA), and RNA quality and concentration was assessed by the BIDMC Molecular Core Facility.

    Construct:

    Article Title: RNA-guided assembly of Rev-RRE nuclear export complexes
    Article Snippet: RNA samples from all constructs were prepared by in vitro transcription using T7 polymerase, with either linearized plasmid or PCR product DNA as templates. .. For SHAPE analysis RNA samples were column-purified using RNA Clean & Concentrator-25 (Zymo Research, Irvine, CA, USA), and for SAXS experiments RNA samples were gel-purified and washed multiple times through filtration.

    IA:

    Article Title: Vaccine Protection Against Zika Virus from Brazil
    Article Snippet: Primers were synthesized by Integrated DNA Technologies (Coralville, IA, USA) and probes by Biosearch Technologies (Petaluma, CA, USA). .. RNA was purified using the RNA clean and concentrator kit (Zymo Research, CA, USA), and RNA quality and concentration was assessed by the BIDMC Molecular Core Facility.

    Mouse Assay:

    Article Title: Histone deacetylase 3 associates with MeCP2 to regulate FOXO and social behavior
    Article Snippet: The CA1 was isolated from the hippocampus of male mice (3-month). .. RNA (3 μg) was DNase I treated (4 U, Worthington Biochemical Corporation), purified using RNA Clean and Concentrator-5 Kit (Zymo Research) according to manufacturers’ instructions and eluted with 14 μl DEPC-treated water.

    Article Title: Vaccine Protection Against Zika Virus from Brazil
    Article Snippet: RNA was purified using the RNA clean and concentrator kit (Zymo Research, CA, USA), and RNA quality and concentration was assessed by the BIDMC Molecular Core Facility. .. RNA was purified using the RNA clean and concentrator kit (Zymo Research, CA, USA), and RNA quality and concentration was assessed by the BIDMC Molecular Core Facility.

    Chromatin Immunoprecipitation:

    Article Title: Multiomics resolution of molecular events during a day in the life of Chlamydomonas
    Article Snippet: RNA was DNase-treated with Turbo DNase (Ambion), followed by a cleaning and concentration step with the RNA Clean & Concentrator-5 kit (Zymo Research). .. RNA was DNase-treated with Turbo DNase (Ambion), followed by a cleaning and concentration step with the RNA Clean & Concentrator-5 kit (Zymo Research).

    Article Title: Impact of CodY protein on metabolism, sporulation and virulence in Clostridioides difficile ribotype 027
    Article Snippet: Only RNA samples with an RNA integrity number > 8 were used for library construction. rRNA was depleted from DNA-free RNA preparations using the RiboZero Magnetic kit (Gram-positive kit; Epicentre). mRNAs were fragmented using the NEB Next RNA Fragmentation Module (New England Biolabs) and further assessed using the Bioanalyzer and RNA Pico Chip. .. Fragmented RNA was purified and concentrated using the RNA Clean & Concentrator-5 kit (Zymo Research Corporation).

    Liquid Chromatography:

    Article Title: Staphylococcus aureus Cas9 is a multiple-turnover enzyme
    Article Snippet: Both S. pyogenes and S. aureus Cas9 were purified at New England Biolabs using standard liquid chromatography protein purification techniques. .. The Zymo RNA Clean & Concentrator-5 kit (#R1016) was purchased from Zymo Research.

    Plasmid Preparation:

    Article Title: RNA-guided assembly of Rev-RRE nuclear export complexes
    Article Snippet: RNA samples from all constructs were prepared by in vitro transcription using T7 polymerase, with either linearized plasmid or PCR product DNA as templates. .. For SHAPE analysis RNA samples were column-purified using RNA Clean & Concentrator-25 (Zymo Research, Irvine, CA, USA), and for SAXS experiments RNA samples were gel-purified and washed multiple times through filtration.

    Software:

    Article Title: Osterix functions downstream of anti-Müllerian hormone signaling to regulate Müllerian duct regression
    Article Snippet: Total RNA was extracted from whole mesonephroi using TRI reagent RT-LS (Molecular Research Center) and RNA Clean and Concentrator- 5 kit (Zymo Research). cDNA was generated from 50 ng of total RNA per biological replicate using the SuperScript II reverse transcriptase (RT) with supplied oligo(dT)12–18 primer (Invitrogen). qPCR was performed on cDNA using a 7900HT Thermocycler (Applied Biosystems) and iTAQ hotstart SYBR Green master mix (Bio-Rad). .. Total RNA was extracted from whole mesonephroi using TRI reagent RT-LS (Molecular Research Center) and RNA Clean and Concentrator- 5 kit (Zymo Research). cDNA was generated from 50 ng of total RNA per biological replicate using the SuperScript II reverse transcriptase (RT) with supplied oligo(dT)12–18 primer (Invitrogen). qPCR was performed on cDNA using a 7900HT Thermocycler (Applied Biosystems) and iTAQ hotstart SYBR Green master mix (Bio-Rad).

    Real-time Polymerase Chain Reaction:

    Article Title: A Synonymous Mutation Upstream of the Gene Encoding a Weak-Link Enzyme Causes an Ultrasensitive Response in Growth Rate
    Article Snippet: An additional DNase treatment performed using Turbo DNase (Life Technologies) according to the manufacturer's protocol was followed by purification with RNA Clean & Concentrator-5 columns (Zymo Research). .. RNA concentration was determined using the Qubit RNA HS assay kit with a Qubit 3.0 fluorometer (Life Technologies).

    Article Title: Osterix functions downstream of anti-Müllerian hormone signaling to regulate Müllerian duct regression
    Article Snippet: Following β-galactosidase staining, reproductive tracts were embedded in paraffin, sectioned at 10 µm, and counterstained with nuclear fast red and/or eosin Y (Sigma Aldrich). .. Total RNA was extracted from whole mesonephroi using TRI reagent RT-LS (Molecular Research Center) and RNA Clean and Concentrator- 5 kit (Zymo Research). cDNA was generated from 50 ng of total RNA per biological replicate using the SuperScript II reverse transcriptase (RT) with supplied oligo(dT)12–18 primer (Invitrogen). qPCR was performed on cDNA using a 7900HT Thermocycler (Applied Biosystems) and iTAQ hotstart SYBR Green master mix (Bio-Rad). .. The fold change of the ΔCT normalized to Actb was used for analysis (2–∆∆Ct method).

    Article Title: H2A.Z controls the stability and mobility of nucleosomes to regulate expression of the LH genes
    Article Snippet: RNA was purified using RNA clean and concentrator-5 kit (Zymo research) with subsequent on-column DNase I (Zymo research) treatment according to the manufacturer's instructions. .. RNA was purified using RNA clean and concentrator-5 kit (Zymo research) with subsequent on-column DNase I (Zymo research) treatment according to the manufacturer's instructions.

    Article Title: mRNA processing in mutant zebrafish lines generated by chemical and CRISPR-mediated mutagenesis produces unexpected transcripts that escape nonsense-mediated decay
    Article Snippet: To genotype individual samples, residual gDNA in the unpurified RNA samples was PCR amplified and sent for Sanger sequencing. .. After genotypes were determined (SnapGene Viewer to view peak trace files), RNA samples were DNAase I-treated and purified (RNA Clean and Concentrator, Zymo Research), served as templates for cDNA synthesis (iScript cDNA Synthesis Kit, Bio-Rad), and ultimately used in qPCR studies to analyze transcript levels. .. qPCR methods included SYBR Green-based methods (Sigma-Aldrich, abca1a , creb3l3a , smyd1a ) and Taqman gene expression assays (ThermoFisher Scientific; cd36 , slc27a2a , and abca1b ). ef1α (for smyd1a ) or 18s rRNA (for all others) levels were used as reference genes.

    Article Title: Diverse electron sources support denitrification under hypoxia in the obligate methanotroph Methylomicrobium album strain BG8
    Article Snippet: The total RNA was then column-purified using RNA clean & concentrator (Zymo Research). .. The total RNA was then column-purified using RNA clean & concentrator (Zymo Research).

    RNA Extraction:

    Article Title: Multiomics resolution of molecular events during a day in the life of Chlamydomonas
    Article Snippet: Paragraph title: RNA Extraction and Library Preparation. ... RNA was DNase-treated with Turbo DNase (Ambion), followed by a cleaning and concentration step with the RNA Clean & Concentrator-5 kit (Zymo Research).

    Article Title: Metabolomic and transcriptomic changes underlying cold and anaerobic stresses after storage of table grapes
    Article Snippet: Paragraph title: RNA extraction and analysis ... The RNA was purified with an RNA Clean and Concentrator Kit (Zymo Research, Irvine, CA, USA).

    Article Title: Telomeric Repeat-Containing RNAs (TERRA) Decrease in Squamous Cell Carcinoma of the Head and Neck Is Associated with Worsened Clinical Outcome
    Article Snippet: Paragraph title: 4.2. RNA Extraction and Purification ... Following each DNase treatment after, the RNA was purified with the RNA Clean and Concentration kit (Zymo Research, Irvine, CA, USA) according to manufacturer’s instruction.

    Article Title: Diverse electron sources support denitrification under hypoxia in the obligate methanotroph Methylomicrobium album strain BG8
    Article Snippet: Paragraph title: RNA Extraction ... The total RNA was then column-purified using RNA clean & concentrator (Zymo Research).

    Selection:

    Article Title: ARM-Seq: AlkB-facilitated RNA methylation sequencing reveals a complex landscape of modified tRNA fragments
    Article Snippet: Paragraph title: Size selection and preparation of RNA sequencing libraries ... RNA was concentrated to 25 µg using RNA Clean and Concentrate-25 (Zymo Research), and 10 µg was treated with DNase I (New England BioLabs).

    Sample Prep:

    Article Title: RNA-guided assembly of Rev-RRE nuclear export complexes
    Article Snippet: Paragraph title: Sample preparation ... For SHAPE analysis RNA samples were column-purified using RNA Clean & Concentrator-25 (Zymo Research, Irvine, CA, USA), and for SAXS experiments RNA samples were gel-purified and washed multiple times through filtration.

    In Vitro:

    Article Title: H2A.Z controls the stability and mobility of nucleosomes to regulate expression of the LH genes
    Article Snippet: Paragraph title: In vitro transcription ... RNA was purified using RNA clean and concentrator-5 kit (Zymo research) with subsequent on-column DNase I (Zymo research) treatment according to the manufacturer's instructions.

    Article Title: RNA-guided assembly of Rev-RRE nuclear export complexes
    Article Snippet: RNA samples from all constructs were prepared by in vitro transcription using T7 polymerase, with either linearized plasmid or PCR product DNA as templates. .. For SHAPE analysis RNA samples were column-purified using RNA Clean & Concentrator-25 (Zymo Research, Irvine, CA, USA), and for SAXS experiments RNA samples were gel-purified and washed multiple times through filtration.

    Next-Generation Sequencing:

    Article Title: Arteriolar niches maintain haematopoietic stem cell quiescence
    Article Snippet: Paragraph title: RNA preparation and next-generation sequencing ... Total RNA from sorted Nesperi and Nesretic cells was extracted using the RNAqueous kit (Life Technologies, Grand Island, NY USA) and concentrated using RNA Clean and Concentrator columns (Zymo Research Corporation, Irvine, CA USA).

    Concentration Assay:

    Article Title: Staphylococcus aureus Cas9 is a multiple-turnover enzyme
    Article Snippet: Protein stock concentration for both Spy- and SauCas9 was measured by absorbance of 280 nm light on a NanoDrop instrument (A280 ) as well as Bio-Rad Bradford assays per manufacturer protocol. .. The Zymo RNA Clean & Concentrator-5 kit (#R1016) was purchased from Zymo Research.

    Article Title: Multiomics resolution of molecular events during a day in the life of Chlamydomonas
    Article Snippet: We extracted total RNA with the TRIzol reagent as previously described ( ). .. RNA was DNase-treated with Turbo DNase (Ambion), followed by a cleaning and concentration step with the RNA Clean & Concentrator-5 kit (Zymo Research). .. RNA quality and concentration were determined on a Nanodrop 2000 (Thermo Fisher Scientific) and an RNA 6000 microfluidic chip on a Bioanalyzer 2100 (Agilent).

    Article Title: Telomeric Repeat-Containing RNAs (TERRA) Decrease in Squamous Cell Carcinoma of the Head and Neck Is Associated with Worsened Clinical Outcome
    Article Snippet: RNA samples were treated twice with 1 U DNase I (Promega, Madison, WI, USA) per μg RNA at 37 °C for 30 min. .. Following each DNase treatment after, the RNA was purified with the RNA Clean and Concentration kit (Zymo Research, Irvine, CA, USA) according to manufacturer’s instruction. .. All samples were diluted in three volumes of denaturation solution (26% formaldehyde, 7% of 20× SSC and 24% formamide) and incubated at 65 °C for 5 min, then, one volume of 20× SSC was added.

    Article Title: Vaccine Protection Against Zika Virus from Brazil
    Article Snippet: The wildtype ZIKV BeH815744 Cap gene was utilized as a standard and was cloned into pcDNA3.1+, and the AmpliCap-Max T7 High Yield Message Maker Kit was used to transcribe RNA (Cellscript, WI, USA). .. RNA was purified using the RNA clean and concentrator kit (Zymo Research, CA, USA), and RNA quality and concentration was assessed by the BIDMC Molecular Core Facility. .. Log dilutions of the RNA standard were reverse transcribed and included with each RT-PCR assay.

    Marker:

    Article Title: ARM-Seq: AlkB-facilitated RNA methylation sequencing reveals a complex landscape of modified tRNA fragments
    Article Snippet: RNA was concentrated to 25 µg using RNA Clean and Concentrate-25 (Zymo Research), and 10 µg was treated with DNase I (New England BioLabs). .. RNA was concentrated to 25 µg using RNA Clean and Concentrate-25 (Zymo Research), and 10 µg was treated with DNase I (New England BioLabs).

    Lysis:

    Article Title: The YTH Domain Protein ECT2 Is an m6A Reader Required for Normal Trichome Branching in Arabidopsis
    Article Snippet: The beads were washed six times with 9 mL of lysis buffer at 4°C. .. The RNA solution was concentrated into a 6 μL volume of RNase-free water using the RNA clean and concentrator kit R1015 from Zymo Research.

    Article Title: Diverse electron sources support denitrification under hypoxia in the obligate methanotroph Methylomicrobium album strain BG8
    Article Snippet: Total nucleic acid was purified according to manufacturer’s instructions with the following modifications: 6 U proteinase K (Qiagen) were added to the cell lysis step and the total precipitated nucleic acid was treated with 30 units of DNase I (Ambion). .. The total RNA was then column-purified using RNA clean & concentrator (Zymo Research).

    Protein Purification:

    Article Title: Staphylococcus aureus Cas9 is a multiple-turnover enzyme
    Article Snippet: Both S. pyogenes and S. aureus Cas9 were purified at New England Biolabs using standard liquid chromatography protein purification techniques. .. The Zymo RNA Clean & Concentrator-5 kit (#R1016) was purchased from Zymo Research.

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    Zymo Research RNA Clean & Concentrator 25
    Sequence relationships between Pol IV/RDR2-dependent <t>RNAs</t> (P4R2 RNAs) and small interfering <t>RNA</t> (siRNAs). ( A ) Correspondence between P4R2 RNA and siRNA loci. P4R2 RNAs were mapped to the Arabidopsis reference genome (TAIR10) and the frequency at which 24 nt siRNAs overlap these P4R2 genomic positions was calculated. To be considered for this analysis, specific siRNAs had to be represented by at least five reads in wild-type Col-0. Supplementary file 2 shows that P4R2 RNA loci include loci confirmed by Li et al., 2015 to generate Pol IV-dependent transcripts. ( B ) P4R2 RNA and 24 nt siRNA spatial relationships. The top panel shows the frequency distribution of P4R2 RNA 5’ end positions relative to siRNA 5’ ends. At position zero on the x-axis, P4R2 RNAs and siRNAs share the same 5’ terminus. Negative values indicate how far (in nucleotides) the 5’ end of the P4R2 RNA is located upstream of an siRNA start position. Likewise, positive values indicate how far the 5’ end of a P4R2 RNA is located downstream of an siRNA start position. The lower panel shows the frequency with which P4R2 RNAs and siRNAs align at 3’ ends. At position zero on the x-axis, P4R2 RNAs and siRNAs share the same 3’ terminus. Negative values occur when P4R2 RNAs end upstream of siRNA 3’ ends, and positive values occur when P4R2 RNAs end downstream of siRNA 3’ ends (computed using FEATnotator, v1.2.2, Podicheti and Mockaitis, 2015 ). DOI: http://dx.doi.org/10.7554/eLife.09591.012
    Rna Clean & Concentrator 25, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sequence relationships between Pol IV/RDR2-dependent RNAs (P4R2 RNAs) and small interfering RNA (siRNAs). ( A ) Correspondence between P4R2 RNA and siRNA loci. P4R2 RNAs were mapped to the Arabidopsis reference genome (TAIR10) and the frequency at which 24 nt siRNAs overlap these P4R2 genomic positions was calculated. To be considered for this analysis, specific siRNAs had to be represented by at least five reads in wild-type Col-0. Supplementary file 2 shows that P4R2 RNA loci include loci confirmed by Li et al., 2015 to generate Pol IV-dependent transcripts. ( B ) P4R2 RNA and 24 nt siRNA spatial relationships. The top panel shows the frequency distribution of P4R2 RNA 5’ end positions relative to siRNA 5’ ends. At position zero on the x-axis, P4R2 RNAs and siRNAs share the same 5’ terminus. Negative values indicate how far (in nucleotides) the 5’ end of the P4R2 RNA is located upstream of an siRNA start position. Likewise, positive values indicate how far the 5’ end of a P4R2 RNA is located downstream of an siRNA start position. The lower panel shows the frequency with which P4R2 RNAs and siRNAs align at 3’ ends. At position zero on the x-axis, P4R2 RNAs and siRNAs share the same 3’ terminus. Negative values occur when P4R2 RNAs end upstream of siRNA 3’ ends, and positive values occur when P4R2 RNAs end downstream of siRNA 3’ ends (computed using FEATnotator, v1.2.2, Podicheti and Mockaitis, 2015 ). DOI: http://dx.doi.org/10.7554/eLife.09591.012

    Journal: eLife

    Article Title: Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis

    doi: 10.7554/eLife.09591

    Figure Lengend Snippet: Sequence relationships between Pol IV/RDR2-dependent RNAs (P4R2 RNAs) and small interfering RNA (siRNAs). ( A ) Correspondence between P4R2 RNA and siRNA loci. P4R2 RNAs were mapped to the Arabidopsis reference genome (TAIR10) and the frequency at which 24 nt siRNAs overlap these P4R2 genomic positions was calculated. To be considered for this analysis, specific siRNAs had to be represented by at least five reads in wild-type Col-0. Supplementary file 2 shows that P4R2 RNA loci include loci confirmed by Li et al., 2015 to generate Pol IV-dependent transcripts. ( B ) P4R2 RNA and 24 nt siRNA spatial relationships. The top panel shows the frequency distribution of P4R2 RNA 5’ end positions relative to siRNA 5’ ends. At position zero on the x-axis, P4R2 RNAs and siRNAs share the same 5’ terminus. Negative values indicate how far (in nucleotides) the 5’ end of the P4R2 RNA is located upstream of an siRNA start position. Likewise, positive values indicate how far the 5’ end of a P4R2 RNA is located downstream of an siRNA start position. The lower panel shows the frequency with which P4R2 RNAs and siRNAs align at 3’ ends. At position zero on the x-axis, P4R2 RNAs and siRNAs share the same 3’ terminus. Negative values occur when P4R2 RNAs end upstream of siRNA 3’ ends, and positive values occur when P4R2 RNAs end downstream of siRNA 3’ ends (computed using FEATnotator, v1.2.2, Podicheti and Mockaitis, 2015 ). DOI: http://dx.doi.org/10.7554/eLife.09591.012

    Article Snippet: The samples were then treated with Terminator exonuclease (Epicentre Biotechnologies, Madison, WI) to remove potentially degraded RNAs with 5’ monophosphates, cleaned with RNA Clean and Concentrator kit (Zymo Research, Irvine, CA), and then treated with RNA 5’ Polyphosphatase (Epicentre Biotechnologies) to convert 5’ triphosphate groups to monophosphates.

    Techniques: Sequencing, Small Interfering RNA

    Pol IV transcripts generated in vitro share features of Pol IV/RDR2-dependent RNAs (P4R2 RNAs) in vivo. ( A and B ) Size and frequency of RNAs transcribed by polymerase (Pol) IV or II in vitro. Pol IV and Pol II were affinity purified by virtue of FLAG epitope tags fused to the C-termini of the NRPD1 or NRPB2 subunits, respectively. In the case of Pol IV, the transgenic NRPD1-FLAG line is null for the endogenous NRPD1 and RDR2 genes, such that Pol IV is free of associated RDR2. Transcripts generated using closed-circular single-stranded M13 virus as the DNA template were subjected to RNA-seq. The frequency and sizes of mapped reads are plotted. ( C and D ) Sequence logos for the 5’ and 3’ ends of Pol IV and II in vitro transcripts. RNA-seq, RNA sequencing. DOI: http://dx.doi.org/10.7554/eLife.09591.015

    Journal: eLife

    Article Title: Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis

    doi: 10.7554/eLife.09591

    Figure Lengend Snippet: Pol IV transcripts generated in vitro share features of Pol IV/RDR2-dependent RNAs (P4R2 RNAs) in vivo. ( A and B ) Size and frequency of RNAs transcribed by polymerase (Pol) IV or II in vitro. Pol IV and Pol II were affinity purified by virtue of FLAG epitope tags fused to the C-termini of the NRPD1 or NRPB2 subunits, respectively. In the case of Pol IV, the transgenic NRPD1-FLAG line is null for the endogenous NRPD1 and RDR2 genes, such that Pol IV is free of associated RDR2. Transcripts generated using closed-circular single-stranded M13 virus as the DNA template were subjected to RNA-seq. The frequency and sizes of mapped reads are plotted. ( C and D ) Sequence logos for the 5’ and 3’ ends of Pol IV and II in vitro transcripts. RNA-seq, RNA sequencing. DOI: http://dx.doi.org/10.7554/eLife.09591.015

    Article Snippet: The samples were then treated with Terminator exonuclease (Epicentre Biotechnologies, Madison, WI) to remove potentially degraded RNAs with 5’ monophosphates, cleaned with RNA Clean and Concentrator kit (Zymo Research, Irvine, CA), and then treated with RNA 5’ Polyphosphatase (Epicentre Biotechnologies) to convert 5’ triphosphate groups to monophosphates.

    Techniques: Generated, In Vitro, In Vivo, Affinity Purification, FLAG-tag, Transgenic Assay, RNA Sequencing Assay, Sequencing

    3’ mismatches detected in 24 nt siRNAs and Pol IV/RDR2-dependent RNAs (P4R2 RNAs) may reflect RDR2 terminal transferase activity. ( A ) Genome browser view of P4R2 RNAs (shades of blue) and 24 nt small interfering RNA (siRNAs) (shades of gray) at a representative locus, an AtSN1 retrotransposon on chromosome 3. Each horizontal bar represents a specific RNA sequence (RNA-seq), with arrows depicting their direction relative to the Arabidopsis reference genome sequence (TAIR10). The intensity of shading reflects the abundance of each RNA species in the RNA-seq dataset. Brightly colored nucleotides, color coded for A, G, C, or U (see inset), represent nucleotides that do not match the corresponding DNA sequence of the locus. The dotted line highlights the coincident 5’ ends of the most abundant P4R2 RNAs at the locus (colored deep purple) and the most abundant siRNAs (colored black). ( B ) Heat map depicting the frequency of mismatched nucleotides at each position of RNAs ranging in size from 15 to 76 nt in dcl2 dcl3 dcl4 triple mutant plants. To correct for the frequency of errors inherent to sequencing, mismatch values for each position of 15–76 nt RNAs in wild-type plants were subtracted prior to plotting the data. Only read sequences with single mismatches or perfect matches to the reference genome were utilized for this analysis. ( C ) Over-expression and purification of recombinant RDR2. The image on the left shows a 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) gel, stained with Coomassie blue, showing molecular weight markers (M), proteins of un-infected High Five cells (lane 1), proteins of High Five cells 72 hr after infection with baculovirus expressing recombinant RNA-dependent RNA polymerase 2 (RDR2) (lane 2), and purified recombinant V5-tagged RDR2 after affinity purification and elution with V5 peptide. The image at right shows anti-RDR2 and anti-V5 immunoblots of the same three protein samples. For RDR2 detection, rabbit anti-RDR2 primary antibody was used in conjunction with donkey anti-rabbit HRP-conjugated secondary antibody. Detection of V5-tagged RDR2 involved anti-V5 HRP conjugate antibody. ( D ) RDR2 terminal transferase activity. Recombinant RDR2 or an active-site mutant form of RDR2 (RDR2-ASM) was incubated with alpha-labeled 32 P-CTP and 51 nt RNA substrates bearing 3’ hydroxyl or 3’ dideoxy termini. Reaction products were subjected to denaturing polyacrylamide gel electrophoresis (PAGE) and autoradiography. For gel lane 4, reaction products were treated with RNase One, which degrades single-stranded RNAs, prior to PAGE. RNA size markers were run in lane M. The 51 nt RNA template, 5’ end-labeled using T4 polynucleotide kinase, was run as a size marker in the lane at far right. DOI: http://dx.doi.org/10.7554/eLife.09591.016

    Journal: eLife

    Article Title: Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis

    doi: 10.7554/eLife.09591

    Figure Lengend Snippet: 3’ mismatches detected in 24 nt siRNAs and Pol IV/RDR2-dependent RNAs (P4R2 RNAs) may reflect RDR2 terminal transferase activity. ( A ) Genome browser view of P4R2 RNAs (shades of blue) and 24 nt small interfering RNA (siRNAs) (shades of gray) at a representative locus, an AtSN1 retrotransposon on chromosome 3. Each horizontal bar represents a specific RNA sequence (RNA-seq), with arrows depicting their direction relative to the Arabidopsis reference genome sequence (TAIR10). The intensity of shading reflects the abundance of each RNA species in the RNA-seq dataset. Brightly colored nucleotides, color coded for A, G, C, or U (see inset), represent nucleotides that do not match the corresponding DNA sequence of the locus. The dotted line highlights the coincident 5’ ends of the most abundant P4R2 RNAs at the locus (colored deep purple) and the most abundant siRNAs (colored black). ( B ) Heat map depicting the frequency of mismatched nucleotides at each position of RNAs ranging in size from 15 to 76 nt in dcl2 dcl3 dcl4 triple mutant plants. To correct for the frequency of errors inherent to sequencing, mismatch values for each position of 15–76 nt RNAs in wild-type plants were subtracted prior to plotting the data. Only read sequences with single mismatches or perfect matches to the reference genome were utilized for this analysis. ( C ) Over-expression and purification of recombinant RDR2. The image on the left shows a 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) gel, stained with Coomassie blue, showing molecular weight markers (M), proteins of un-infected High Five cells (lane 1), proteins of High Five cells 72 hr after infection with baculovirus expressing recombinant RNA-dependent RNA polymerase 2 (RDR2) (lane 2), and purified recombinant V5-tagged RDR2 after affinity purification and elution with V5 peptide. The image at right shows anti-RDR2 and anti-V5 immunoblots of the same three protein samples. For RDR2 detection, rabbit anti-RDR2 primary antibody was used in conjunction with donkey anti-rabbit HRP-conjugated secondary antibody. Detection of V5-tagged RDR2 involved anti-V5 HRP conjugate antibody. ( D ) RDR2 terminal transferase activity. Recombinant RDR2 or an active-site mutant form of RDR2 (RDR2-ASM) was incubated with alpha-labeled 32 P-CTP and 51 nt RNA substrates bearing 3’ hydroxyl or 3’ dideoxy termini. Reaction products were subjected to denaturing polyacrylamide gel electrophoresis (PAGE) and autoradiography. For gel lane 4, reaction products were treated with RNase One, which degrades single-stranded RNAs, prior to PAGE. RNA size markers were run in lane M. The 51 nt RNA template, 5’ end-labeled using T4 polynucleotide kinase, was run as a size marker in the lane at far right. DOI: http://dx.doi.org/10.7554/eLife.09591.016

    Article Snippet: The samples were then treated with Terminator exonuclease (Epicentre Biotechnologies, Madison, WI) to remove potentially degraded RNAs with 5’ monophosphates, cleaned with RNA Clean and Concentrator kit (Zymo Research, Irvine, CA), and then treated with RNA 5’ Polyphosphatase (Epicentre Biotechnologies) to convert 5’ triphosphate groups to monophosphates.

    Techniques: Activity Assay, Small Interfering RNA, Sequencing, RNA Sequencing Assay, Mutagenesis, Over Expression, Purification, Recombinant, Electrophoresis, Polyacrylamide Gel Electrophoresis, Staining, Molecular Weight, Infection, Expressing, Affinity Purification, Western Blot, Incubation, Labeling, Autoradiography, Marker

    RNA blot analyses of 24 nt siRNAs and their precursors. ( A ) The small RNA blot was successively hybridized to probes representing either strand of the siR1003 duplex, a small interfering RNA (siRNA) that is derived from intergenic regions separating 5S ribosomal RNA (rRNA) gene repeats (top two images), as well as to a trans -acting siRNA (ta-siR255) and a microRNA (miR160) probe. An image of the stained gel under fluorescent illumination (in the region that includes 5S rRNA and transfer RNAs [tRNAs]) is shown at the bottom as a loading control. ( B ) RNA blot of small RNAs isolated from wild-type (ecotype Col-0) or dcl2 dcl3 dcl4 triple mutant ( dcl2/3/4 ) plants, with or without prior treatment with ribonuclease V1 or ribonuclease A. The blot was hybridized to a probe designed to detect siR1003 ‘sense’, which arises from 5S rRNA gene intergenic spacers. ( C ) Dicing of precursor RNAs by DICER-like 3 (DCL3) in vitro. RNA isolated from wild-type (ecotype Col-0) or from dcl2/3/4 triple mutant plants was incubated with anti-FLAG resin that had been incubated with protein extraction buffer, a cell-free extract of wild-type (Col-0) plants, or a cell-free extract of transgenic plants expressing FLAG-tagged DCL3. RNAs were then purified, subjected to blotting and hybridized to the siR1003 ‘sense’ probe. DOI: http://dx.doi.org/10.7554/eLife.09591.004

    Journal: eLife

    Article Title: Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis

    doi: 10.7554/eLife.09591

    Figure Lengend Snippet: RNA blot analyses of 24 nt siRNAs and their precursors. ( A ) The small RNA blot was successively hybridized to probes representing either strand of the siR1003 duplex, a small interfering RNA (siRNA) that is derived from intergenic regions separating 5S ribosomal RNA (rRNA) gene repeats (top two images), as well as to a trans -acting siRNA (ta-siR255) and a microRNA (miR160) probe. An image of the stained gel under fluorescent illumination (in the region that includes 5S rRNA and transfer RNAs [tRNAs]) is shown at the bottom as a loading control. ( B ) RNA blot of small RNAs isolated from wild-type (ecotype Col-0) or dcl2 dcl3 dcl4 triple mutant ( dcl2/3/4 ) plants, with or without prior treatment with ribonuclease V1 or ribonuclease A. The blot was hybridized to a probe designed to detect siR1003 ‘sense’, which arises from 5S rRNA gene intergenic spacers. ( C ) Dicing of precursor RNAs by DICER-like 3 (DCL3) in vitro. RNA isolated from wild-type (ecotype Col-0) or from dcl2/3/4 triple mutant plants was incubated with anti-FLAG resin that had been incubated with protein extraction buffer, a cell-free extract of wild-type (Col-0) plants, or a cell-free extract of transgenic plants expressing FLAG-tagged DCL3. RNAs were then purified, subjected to blotting and hybridized to the siR1003 ‘sense’ probe. DOI: http://dx.doi.org/10.7554/eLife.09591.004

    Article Snippet: The samples were then treated with Terminator exonuclease (Epicentre Biotechnologies, Madison, WI) to remove potentially degraded RNAs with 5’ monophosphates, cleaned with RNA Clean and Concentrator kit (Zymo Research, Irvine, CA), and then treated with RNA 5’ Polyphosphatase (Epicentre Biotechnologies) to convert 5’ triphosphate groups to monophosphates.

    Techniques: Northern blot, Small Interfering RNA, Derivative Assay, Staining, Isolation, Mutagenesis, In Vitro, Incubation, Protein Extraction, Transgenic Assay, Expressing, Purification

    P4R2 RNAs are co-dependent on Pol IV and RDR2. ( A–C ) Browser views of three additional 24 nt small interfering RNA (siRNA) loci at which Pol IV/RDR2-dependent RNAs (P4R2 RNAs) that accumulate in dcl2 dcl3 dcl4 ( dcl2/3/4 ) mutants are also observed in wild-type (Col-0) plants (with five or more reads for at least one P4R2 species) but are not observed in nrpd1 polymerase IV (Pol IV) or rdr2 mutants. DOI: http://dx.doi.org/10.7554/eLife.09591.011

    Journal: eLife

    Article Title: Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis

    doi: 10.7554/eLife.09591

    Figure Lengend Snippet: P4R2 RNAs are co-dependent on Pol IV and RDR2. ( A–C ) Browser views of three additional 24 nt small interfering RNA (siRNA) loci at which Pol IV/RDR2-dependent RNAs (P4R2 RNAs) that accumulate in dcl2 dcl3 dcl4 ( dcl2/3/4 ) mutants are also observed in wild-type (Col-0) plants (with five or more reads for at least one P4R2 species) but are not observed in nrpd1 polymerase IV (Pol IV) or rdr2 mutants. DOI: http://dx.doi.org/10.7554/eLife.09591.011

    Article Snippet: The samples were then treated with Terminator exonuclease (Epicentre Biotechnologies, Madison, WI) to remove potentially degraded RNAs with 5’ monophosphates, cleaned with RNA Clean and Concentrator kit (Zymo Research, Irvine, CA), and then treated with RNA 5’ Polyphosphatase (Epicentre Biotechnologies) to convert 5’ triphosphate groups to monophosphates.

    Techniques: Small Interfering RNA

    Browser view of Pol IV/RDR2-dependent RNAs (P4R2 RNAs) and 24/23 nt small interfering RNA (siRNAs) in the intergenic spacer region of a 5S ribosomal RNA (rRNA) gene repeat unit. An isolated 5S rRNA gene repeat (∼500 bp, gray horizontal bar with red transcript region) is shown within its 5 kb chromosomal context, flanked by two transposable elements, shown in yellow, and a pseudogene, shown in blue. Below the diagram, P4R2 RNAs are depicted as horizontal bars shown in shades of blue whereas 24 and 23 nt siRNAs are shown in shades of gray to black, with color intensity reflecting abundance (read counts are provided for several examples). Each bar represents a specific RNA sequence, with arrows depicting the RNA strand orientation relative to the reference genome sequence (TAIR10). Dotted vertical lines provide alignments and show that the ends of highly abundant ( > 100 reads) siRNA species tend to coincide with the ends of P4R2 RNAs for which there is more than a single read. DOI: http://dx.doi.org/10.7554/eLife.09591.009

    Journal: eLife

    Article Title: Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis

    doi: 10.7554/eLife.09591

    Figure Lengend Snippet: Browser view of Pol IV/RDR2-dependent RNAs (P4R2 RNAs) and 24/23 nt small interfering RNA (siRNAs) in the intergenic spacer region of a 5S ribosomal RNA (rRNA) gene repeat unit. An isolated 5S rRNA gene repeat (∼500 bp, gray horizontal bar with red transcript region) is shown within its 5 kb chromosomal context, flanked by two transposable elements, shown in yellow, and a pseudogene, shown in blue. Below the diagram, P4R2 RNAs are depicted as horizontal bars shown in shades of blue whereas 24 and 23 nt siRNAs are shown in shades of gray to black, with color intensity reflecting abundance (read counts are provided for several examples). Each bar represents a specific RNA sequence, with arrows depicting the RNA strand orientation relative to the reference genome sequence (TAIR10). Dotted vertical lines provide alignments and show that the ends of highly abundant ( > 100 reads) siRNA species tend to coincide with the ends of P4R2 RNAs for which there is more than a single read. DOI: http://dx.doi.org/10.7554/eLife.09591.009

    Article Snippet: The samples were then treated with Terminator exonuclease (Epicentre Biotechnologies, Madison, WI) to remove potentially degraded RNAs with 5’ monophosphates, cleaned with RNA Clean and Concentrator kit (Zymo Research, Irvine, CA), and then treated with RNA 5’ Polyphosphatase (Epicentre Biotechnologies) to convert 5’ triphosphate groups to monophosphates.

    Techniques: Small Interfering RNA, Isolation, Sequencing

    Number of unique sequences among RNAs of 15–94 nt in wild-type or dcl2 dcl3 dcl4 ( dcl2/3/4/) triple mutants. RNAs representing a specific sequence and a particular strand polarity were counted only once, regardless of the actual abundance of RNA sequencing (RNA-seq) reads for that sequence in the dataset. RNA-seq counts for wild-type are shown in blue. RNA-seq counts for dcl2 dcl3 dcl4 mutants are shown in orange. Overlapping read counts are shown in deep red. DOI: http://dx.doi.org/10.7554/eLife.09591.008

    Journal: eLife

    Article Title: Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis

    doi: 10.7554/eLife.09591

    Figure Lengend Snippet: Number of unique sequences among RNAs of 15–94 nt in wild-type or dcl2 dcl3 dcl4 ( dcl2/3/4/) triple mutants. RNAs representing a specific sequence and a particular strand polarity were counted only once, regardless of the actual abundance of RNA sequencing (RNA-seq) reads for that sequence in the dataset. RNA-seq counts for wild-type are shown in blue. RNA-seq counts for dcl2 dcl3 dcl4 mutants are shown in orange. Overlapping read counts are shown in deep red. DOI: http://dx.doi.org/10.7554/eLife.09591.008

    Article Snippet: The samples were then treated with Terminator exonuclease (Epicentre Biotechnologies, Madison, WI) to remove potentially degraded RNAs with 5’ monophosphates, cleaned with RNA Clean and Concentrator kit (Zymo Research, Irvine, CA), and then treated with RNA 5’ Polyphosphatase (Epicentre Biotechnologies) to convert 5’ triphosphate groups to monophosphates.

    Techniques: Sequencing, RNA Sequencing Assay

    Biogenesis of 24 nt siRNAs and their role in RNA-directed DNA methylation. A simplified cartoon of the RNA-directed DNA methylation pathway. Polymerase (Pol) IV and RNA-dependent RNA polymerase (RDR2) physically associate and are required for the synthesis of double-stranded RNAs (dsRNA) that are diced by DICER-like 3 (DCL3) into 24 nt siRNA duplexes. Upon loading into Argonaute 4 (AGO4), the siRNA-AGO4 complex finds its target sites by binding to Pol V transcripts and by interacting with the C-terminal domain (CTD) of the Pol V largest subunit. The cytosine methyltransferase DRM2 is ultimately recruited to Pol V-transcribed loci, resulting in de novo cytosine methylation in all sequence contexts (CG, CHG and CHH; where H represents a nucleotide other than G). DOI: http://dx.doi.org/10.7554/eLife.09591.003

    Journal: eLife

    Article Title: Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis

    doi: 10.7554/eLife.09591

    Figure Lengend Snippet: Biogenesis of 24 nt siRNAs and their role in RNA-directed DNA methylation. A simplified cartoon of the RNA-directed DNA methylation pathway. Polymerase (Pol) IV and RNA-dependent RNA polymerase (RDR2) physically associate and are required for the synthesis of double-stranded RNAs (dsRNA) that are diced by DICER-like 3 (DCL3) into 24 nt siRNA duplexes. Upon loading into Argonaute 4 (AGO4), the siRNA-AGO4 complex finds its target sites by binding to Pol V transcripts and by interacting with the C-terminal domain (CTD) of the Pol V largest subunit. The cytosine methyltransferase DRM2 is ultimately recruited to Pol V-transcribed loci, resulting in de novo cytosine methylation in all sequence contexts (CG, CHG and CHH; where H represents a nucleotide other than G). DOI: http://dx.doi.org/10.7554/eLife.09591.003

    Article Snippet: The samples were then treated with Terminator exonuclease (Epicentre Biotechnologies, Madison, WI) to remove potentially degraded RNAs with 5’ monophosphates, cleaned with RNA Clean and Concentrator kit (Zymo Research, Irvine, CA), and then treated with RNA 5’ Polyphosphatase (Epicentre Biotechnologies) to convert 5’ triphosphate groups to monophosphates.

    Techniques: DNA Methylation Assay, Binding Assay, Methylation, Sequencing

    Relative composition of the OMVs RNA cargo for each culture condition. Individual colored bars represent the relative amount of each RNA class in the respective OMV RNA-Seq data (average of the biological triplicates). Number of reads have been normalized with DESeq2 . RNA classes are defined from the Salmonella LT2 genome annotation (NCBI accession number AE006468.2 ) or pSLT plasmid annotation (NCBI accession number AE006471.2 ), with supplemental small RNA annotations from Srikumar et al. (2015) . The existence of significant proportion differences among conditions for a specific RNA class was assessed by one-way ANOVA and F -test. Significant differences for rRNA and mRNA classes between conditions are indicated under the graph, determined by Tukey’s range test. Further details for other RNA classes can be found in Supplementary Dataset B .

    Journal: Frontiers in Microbiology

    Article Title: The RNA Complement of Outer Membrane Vesicles From Salmonella enterica Serovar Typhimurium Under Distinct Culture Conditions

    doi: 10.3389/fmicb.2018.02015

    Figure Lengend Snippet: Relative composition of the OMVs RNA cargo for each culture condition. Individual colored bars represent the relative amount of each RNA class in the respective OMV RNA-Seq data (average of the biological triplicates). Number of reads have been normalized with DESeq2 . RNA classes are defined from the Salmonella LT2 genome annotation (NCBI accession number AE006468.2 ) or pSLT plasmid annotation (NCBI accession number AE006471.2 ), with supplemental small RNA annotations from Srikumar et al. (2015) . The existence of significant proportion differences among conditions for a specific RNA class was assessed by one-way ANOVA and F -test. Significant differences for rRNA and mRNA classes between conditions are indicated under the graph, determined by Tukey’s range test. Further details for other RNA classes can be found in Supplementary Dataset B .

    Article Snippet: OMV-associated RNA was further concentrated using a Zymo Research RNA clean up and concentrator kit (including On-column DNaseI treatment) up to a volume of 6 μl (corresponding to 1.5 l of original culture).

    Techniques: RNA Sequencing Assay, Plasmid Preparation

    Repartition of enriched RNAs in/on OMVs isolated from the different analyzed in vitro culture conditions. UpSet plot representing the number of enriched RNAs shared between different conditions. One unit represents one expressed gene. The graph was generated using the UpSet R package. Enriched RNAs were determined using DESeq2 , calculating differential presence between intracellular and OMV-associated fractions for each condition. Expressed genes with an average number of reads over 5 for a given set of triplicates, a log 2 Fold Change over 2, and a corresponding adjusted p -value under 0.05 were selected. ncRNA class attribution is defined from the Salmonella LT2 genome annotation (NCBI accession number AE006468.2 ) or pSLT plasmid annotation (NCBI accession number AE006471.2 ). Moreover, small RNA annotations from Srikumar et al. (2015) were mapped to the LT2 genome. Only sequences that had 100% sequence identity over the full length on LT2 strain were kept. Identical datasets were used for the generation of Figure 8 and Supplementary Figure S6B .

    Journal: Frontiers in Microbiology

    Article Title: The RNA Complement of Outer Membrane Vesicles From Salmonella enterica Serovar Typhimurium Under Distinct Culture Conditions

    doi: 10.3389/fmicb.2018.02015

    Figure Lengend Snippet: Repartition of enriched RNAs in/on OMVs isolated from the different analyzed in vitro culture conditions. UpSet plot representing the number of enriched RNAs shared between different conditions. One unit represents one expressed gene. The graph was generated using the UpSet R package. Enriched RNAs were determined using DESeq2 , calculating differential presence between intracellular and OMV-associated fractions for each condition. Expressed genes with an average number of reads over 5 for a given set of triplicates, a log 2 Fold Change over 2, and a corresponding adjusted p -value under 0.05 were selected. ncRNA class attribution is defined from the Salmonella LT2 genome annotation (NCBI accession number AE006468.2 ) or pSLT plasmid annotation (NCBI accession number AE006471.2 ). Moreover, small RNA annotations from Srikumar et al. (2015) were mapped to the LT2 genome. Only sequences that had 100% sequence identity over the full length on LT2 strain were kept. Identical datasets were used for the generation of Figure 8 and Supplementary Figure S6B .

    Article Snippet: OMV-associated RNA was further concentrated using a Zymo Research RNA clean up and concentrator kit (including On-column DNaseI treatment) up to a volume of 6 μl (corresponding to 1.5 l of original culture).

    Techniques: Isolation, In Vitro, Generated, Plasmid Preparation, Sequencing

    Visualization of intracellular and OMV-related read coverage plots of distinct sRNAs in distinct culture conditions. RNA coverage from sequencing data was visualized with the Integrative Genomics Viewer software (v2.4.8) using default parameters ( Robinson et al., 2011 ). Each plot represents the raw number of reads mapped along the observed sequence, automatically scaled relatively to the highest number of read existing in this portion of the genome. The three plots in dark colors at the top of each window represents the sequencing coverage of OMV-associated fracti ons, in biological triplicate for each condition. On the contrary, the three plots in lighter colors at the bottom of each window show the coverage for the same RNA but from the corresponding intracellular fractions. The arrows under each window precise the genes positions and orientations, according to the data extracted from the Salmonella LT2 genome annotation (NCBI accession number AE006468.2 ) or pSLT plasmid annotation (NCBI accession number AE006471.2 ). The identical coverage patterns observed in both fractions for the displayed genes stand for a native export through OMVs without specific processing or degradation.

    Journal: Frontiers in Microbiology

    Article Title: The RNA Complement of Outer Membrane Vesicles From Salmonella enterica Serovar Typhimurium Under Distinct Culture Conditions

    doi: 10.3389/fmicb.2018.02015

    Figure Lengend Snippet: Visualization of intracellular and OMV-related read coverage plots of distinct sRNAs in distinct culture conditions. RNA coverage from sequencing data was visualized with the Integrative Genomics Viewer software (v2.4.8) using default parameters ( Robinson et al., 2011 ). Each plot represents the raw number of reads mapped along the observed sequence, automatically scaled relatively to the highest number of read existing in this portion of the genome. The three plots in dark colors at the top of each window represents the sequencing coverage of OMV-associated fracti ons, in biological triplicate for each condition. On the contrary, the three plots in lighter colors at the bottom of each window show the coverage for the same RNA but from the corresponding intracellular fractions. The arrows under each window precise the genes positions and orientations, according to the data extracted from the Salmonella LT2 genome annotation (NCBI accession number AE006468.2 ) or pSLT plasmid annotation (NCBI accession number AE006471.2 ). The identical coverage patterns observed in both fractions for the displayed genes stand for a native export through OMVs without specific processing or degradation.

    Article Snippet: OMV-associated RNA was further concentrated using a Zymo Research RNA clean up and concentrator kit (including On-column DNaseI treatment) up to a volume of 6 μl (corresponding to 1.5 l of original culture).

    Techniques: Sequencing, Software, Plasmid Preparation

    RNase protection assay of selected protein-associated sRNAs isolated from OMVs. OMV isolation and enzymatic digestions were performed from SPI-1ind condition as described in Section “Materials and Methods (see section “OMV Purification and RNA Extraction”).” Crude OMV preparation was divided in four fractions. One was kept untouched (Untreated). The second was subjected to the same protocol than the third and fourth fraction without any enzyme added (Control). The third was digested by RNaseA. The fourth was digested by ProteinaseK followed by proteinase inactivation and RNAseA digestion. The protocol was then followed as described in Section “OMV Purification and RNA Extraction” until cDNA libraries from OMV-associated RNAs were obtained End-point PCR was then conducted with the same primers and conditions used previously, for SsrS , CsrC , 10Sa , and rnpB genes. Resulting samples were deposited on a 3% w/v agarose gel in TBE buffer, subjected to electrophoresis and stained with Ethidium Bromide. Most of the signal is still present after enzymatic digestion, showing that the RNAs are protected by the vesicle membrane. –RT controls and positive controls for enzymatic digestions can be seen in Supplementary Figure S5 .

    Journal: Frontiers in Microbiology

    Article Title: The RNA Complement of Outer Membrane Vesicles From Salmonella enterica Serovar Typhimurium Under Distinct Culture Conditions

    doi: 10.3389/fmicb.2018.02015

    Figure Lengend Snippet: RNase protection assay of selected protein-associated sRNAs isolated from OMVs. OMV isolation and enzymatic digestions were performed from SPI-1ind condition as described in Section “Materials and Methods (see section “OMV Purification and RNA Extraction”).” Crude OMV preparation was divided in four fractions. One was kept untouched (Untreated). The second was subjected to the same protocol than the third and fourth fraction without any enzyme added (Control). The third was digested by RNaseA. The fourth was digested by ProteinaseK followed by proteinase inactivation and RNAseA digestion. The protocol was then followed as described in Section “OMV Purification and RNA Extraction” until cDNA libraries from OMV-associated RNAs were obtained End-point PCR was then conducted with the same primers and conditions used previously, for SsrS , CsrC , 10Sa , and rnpB genes. Resulting samples were deposited on a 3% w/v agarose gel in TBE buffer, subjected to electrophoresis and stained with Ethidium Bromide. Most of the signal is still present after enzymatic digestion, showing that the RNAs are protected by the vesicle membrane. –RT controls and positive controls for enzymatic digestions can be seen in Supplementary Figure S5 .

    Article Snippet: OMV-associated RNA was further concentrated using a Zymo Research RNA clean up and concentrator kit (including On-column DNaseI treatment) up to a volume of 6 μl (corresponding to 1.5 l of original culture).

    Techniques: Rnase Protection Assay, Isolation, Purification, RNA Extraction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis, Staining

    Differential abundance of RNAs in OMVs and their intracellular counterparts. (A–E) Scatterplots showing the reads repartition within a given growth condition. Each dot represents the relative abundance of an expressed gene in or attached to the OMVs (vertical axis) and in the intracellular (horizontal axis) fraction for each growth condition used in this study. RNA annotation from the Salmonella LT2 genome (NCBI accession number AE006468.2 ) or pSLT plasmid (NCBI accession number AE006471.2 ) is precized by a color code. Number of reads were normalized by sum normalization for each condition using the decostand function from the vegan R package. Ribosomal RNAs, which were experimentally depleted for intracellular fractions, and removed in silico for OMV fractions, are omitted. (F) Color legend used to underline the RNA class of each annotated transcript in the graphs.

    Journal: Frontiers in Microbiology

    Article Title: The RNA Complement of Outer Membrane Vesicles From Salmonella enterica Serovar Typhimurium Under Distinct Culture Conditions

    doi: 10.3389/fmicb.2018.02015

    Figure Lengend Snippet: Differential abundance of RNAs in OMVs and their intracellular counterparts. (A–E) Scatterplots showing the reads repartition within a given growth condition. Each dot represents the relative abundance of an expressed gene in or attached to the OMVs (vertical axis) and in the intracellular (horizontal axis) fraction for each growth condition used in this study. RNA annotation from the Salmonella LT2 genome (NCBI accession number AE006468.2 ) or pSLT plasmid (NCBI accession number AE006471.2 ) is precized by a color code. Number of reads were normalized by sum normalization for each condition using the decostand function from the vegan R package. Ribosomal RNAs, which were experimentally depleted for intracellular fractions, and removed in silico for OMV fractions, are omitted. (F) Color legend used to underline the RNA class of each annotated transcript in the graphs.

    Article Snippet: OMV-associated RNA was further concentrated using a Zymo Research RNA clean up and concentrator kit (including On-column DNaseI treatment) up to a volume of 6 μl (corresponding to 1.5 l of original culture).

    Techniques: Plasmid Preparation, In Silico