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    T4 RNA Ligase 1 ssRNA Ligase
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    T4 RNA Ligase 1 ssRNA Ligase 5 000 units
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    m0204l
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    5 000 units
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    RNA Ligases
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    New England Biolabs dna free rna
    T4 RNA Ligase 1 ssRNA Ligase
    T4 RNA Ligase 1 ssRNA Ligase 5 000 units
    https://www.bioz.com/result/dna free rna/product/New England Biolabs
    Average 93 stars, based on 1270 article reviews
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    dna free rna - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "A conserved RpoS-dependent small RNA controls the synthesis of major porin OmpD"

    Article Title: A conserved RpoS-dependent small RNA controls the synthesis of major porin OmpD

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1156

    ( A ) Regulation of OmpD-GFP reporter fusions by SdsR. Salmonella ΔsdsR ΔompD cells carrying the control vector or pP L -SdsR were co-transformed with low-copy plasmids expressing gfp alone or a series of translational ompD::gfp fusions spanning the complete 5′-UTR plus an increasing number of nucleotides of the ompD coding sequence (D+3:: gfp ; D+45:: gfp ; D+78:: gfp ; D+99:: gfp ; see Supplementary Table S2 for details on plasmids) as depicted in (D). Whole-protein samples were collected from cells grown to an OD 600 of 2.0, and regulation of reporter fusions was determined by signal quantification on western blots. Relative GFP levels in the presence of the control plasmid (black bars; set to 100) or the constitutive pP L -SdsR (grey bars); errors indicate standard deviation from three biological replicates. ( B ) Schematic illustration of the 3′-RACE approach employed for target site determination. ( C ) 3′-RACE analysis of ompD mRNA fragments enriched upon SdsR pulse expression. cDNA was prepared from total RNA of Δ sdsR cells as well as the Δ sdsR Δ ompD control strain prior to and at indicated timepoints after SdsR induction from an inducible P BAD promoter. Salmonella genomic DNA (gDNA) served as a control template. DNA fragments were recovered from the indicated band of ∼150 bp (lane 6), and ompD 3′-ends were determined by sequencing of subcloned fragments. ( D ) Location of ompD 3′-ends obtained by 3′-RACE analysis. The ompD :: gfp reporter plasmids and their regulation by SdsR (see Figure 5 A) are represented schematically. The filled circle indicates the approximate coverage of ompD mRNA by the 30S ribosomal subunit binding to the RBS. Position as well as frequency of enriched break-down products determined by 3′-RACE ( Figure 5 C) are shown below the ompD CDS.
    Figure Legend Snippet: ( A ) Regulation of OmpD-GFP reporter fusions by SdsR. Salmonella ΔsdsR ΔompD cells carrying the control vector or pP L -SdsR were co-transformed with low-copy plasmids expressing gfp alone or a series of translational ompD::gfp fusions spanning the complete 5′-UTR plus an increasing number of nucleotides of the ompD coding sequence (D+3:: gfp ; D+45:: gfp ; D+78:: gfp ; D+99:: gfp ; see Supplementary Table S2 for details on plasmids) as depicted in (D). Whole-protein samples were collected from cells grown to an OD 600 of 2.0, and regulation of reporter fusions was determined by signal quantification on western blots. Relative GFP levels in the presence of the control plasmid (black bars; set to 100) or the constitutive pP L -SdsR (grey bars); errors indicate standard deviation from three biological replicates. ( B ) Schematic illustration of the 3′-RACE approach employed for target site determination. ( C ) 3′-RACE analysis of ompD mRNA fragments enriched upon SdsR pulse expression. cDNA was prepared from total RNA of Δ sdsR cells as well as the Δ sdsR Δ ompD control strain prior to and at indicated timepoints after SdsR induction from an inducible P BAD promoter. Salmonella genomic DNA (gDNA) served as a control template. DNA fragments were recovered from the indicated band of ∼150 bp (lane 6), and ompD 3′-ends were determined by sequencing of subcloned fragments. ( D ) Location of ompD 3′-ends obtained by 3′-RACE analysis. The ompD :: gfp reporter plasmids and their regulation by SdsR (see Figure 5 A) are represented schematically. The filled circle indicates the approximate coverage of ompD mRNA by the 30S ribosomal subunit binding to the RBS. Position as well as frequency of enriched break-down products determined by 3′-RACE ( Figure 5 C) are shown below the ompD CDS.

    Techniques Used: Plasmid Preparation, Transformation Assay, Expressing, Sequencing, Western Blot, Standard Deviation, Binding Assay

    2) Product Images from "Efficient synthesis of stably adenylated DNA and RNA adapters for microRNA capture using T4 RNA ligase 1"

    Article Title: Efficient synthesis of stably adenylated DNA and RNA adapters for microRNA capture using T4 RNA ligase 1

    Journal: Scientific Reports

    doi: 10.1038/srep15620

    ( a ) Schematic illustration of the high efficiency, purification- and template-free, adapter oligonucleotide adenylation method using T4 RNA ligase 1. The 3′ end of the adapter oligo was blocked by –ddC modification to prevent circularization and concatemerization. The 5′ base (shown in black) was swapped between dA, dC, dG, dT, rA, rC, rG, and rU to test bias. ( b ) The adapter adenylation efficiency was investigated as a function of 5′ terminal nucleotide. The reaction conditions were modified to exaggerate differences in efficiency (10 μL volume, 100 units ligase per nanomole adapter, 0.1 nanomole adapter, 30% PEG, 1 hour incubation). The rC and dG adapters are the most and least efficiently adenylated, respectively. ( c ) The adapter adenylation efficiency was then measured as a function of PEG % for a few representative adapters. In all cases, efficiency monotonically increased with PEG %. ( d ) Comparison of adenylation efficiency of as a function of PEG % under standard reaction conditions using the rA and dA adapters. Both the dA and rA adapters are efficiently adenylated at 35% PEG.
    Figure Legend Snippet: ( a ) Schematic illustration of the high efficiency, purification- and template-free, adapter oligonucleotide adenylation method using T4 RNA ligase 1. The 3′ end of the adapter oligo was blocked by –ddC modification to prevent circularization and concatemerization. The 5′ base (shown in black) was swapped between dA, dC, dG, dT, rA, rC, rG, and rU to test bias. ( b ) The adapter adenylation efficiency was investigated as a function of 5′ terminal nucleotide. The reaction conditions were modified to exaggerate differences in efficiency (10 μL volume, 100 units ligase per nanomole adapter, 0.1 nanomole adapter, 30% PEG, 1 hour incubation). The rC and dG adapters are the most and least efficiently adenylated, respectively. ( c ) The adapter adenylation efficiency was then measured as a function of PEG % for a few representative adapters. In all cases, efficiency monotonically increased with PEG %. ( d ) Comparison of adenylation efficiency of as a function of PEG % under standard reaction conditions using the rA and dA adapters. Both the dA and rA adapters are efficiently adenylated at 35% PEG.

    Techniques Used: Purification, Modification, Incubation

    microRNA-adapter ligation was performed using adenylated adapters generated by either (a) T4 RNA ligase 1 or (c) archaeal RNA ligase. The adapters were labeled with Cy5 while the synthetic microRNA were labeled with Cy3. Lanes 1 and 2 show that both methods are capable of fully adenylating the adapters. Lanes 4 and 6 show that let-7a microRNA can be effectively ligated both in the absence and presence of total RNA background. Lane 5 shows that large RNA molecules within the total RNA are captured by both adapters. No de-adenylation is observed with either method. ( b ) The T4 RNA ligase 1 adenylated adapter was used to capture RNA from 10, 100, or 1000 ng of pancreatic tissue total RNA spiked with 0.01 picomoles of 6 synthetic microRNA. The three ligation products from the top are large RNA molecules intrinsic to the total RNA that have been captured by the adapter. As expected, they vary in linear proportion to the total RNA input. The band in the middle is the spiked microRNA captured by the adapter which remains constant across all three samples as expected. The large band at the bottom of the gel is free adenylated Cy5 adapter.
    Figure Legend Snippet: microRNA-adapter ligation was performed using adenylated adapters generated by either (a) T4 RNA ligase 1 or (c) archaeal RNA ligase. The adapters were labeled with Cy5 while the synthetic microRNA were labeled with Cy3. Lanes 1 and 2 show that both methods are capable of fully adenylating the adapters. Lanes 4 and 6 show that let-7a microRNA can be effectively ligated both in the absence and presence of total RNA background. Lane 5 shows that large RNA molecules within the total RNA are captured by both adapters. No de-adenylation is observed with either method. ( b ) The T4 RNA ligase 1 adenylated adapter was used to capture RNA from 10, 100, or 1000 ng of pancreatic tissue total RNA spiked with 0.01 picomoles of 6 synthetic microRNA. The three ligation products from the top are large RNA molecules intrinsic to the total RNA that have been captured by the adapter. As expected, they vary in linear proportion to the total RNA input. The band in the middle is the spiked microRNA captured by the adapter which remains constant across all three samples as expected. The large band at the bottom of the gel is free adenylated Cy5 adapter.

    Techniques Used: Ligation, Generated, Labeling

    Adenylated adapters generated using either T4 RNA ligase 1 or archaeal RNA ligase were used for microRNA-adapter ligation of a mixture containing 80 nt let-7a precursor DNA molecules and 22 nt let-7a mature microRNA molecules. The amount of PEG in the reaction mixture was also varied. Circularized DNA ligation product is only generated using the archaeal RNA ligase adenylated adapters.
    Figure Legend Snippet: Adenylated adapters generated using either T4 RNA ligase 1 or archaeal RNA ligase were used for microRNA-adapter ligation of a mixture containing 80 nt let-7a precursor DNA molecules and 22 nt let-7a mature microRNA molecules. The amount of PEG in the reaction mixture was also varied. Circularized DNA ligation product is only generated using the archaeal RNA ligase adenylated adapters.

    Techniques Used: Generated, Ligation, DNA Ligation

    3) Product Images from "Detecting RNA-RNA interactions in E. coli using a modified CLASH method"

    Article Title: Detecting RNA-RNA interactions in E. coli using a modified CLASH method

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-3725-3

    Schematic overview of the modified protocol. a , wet experiment. Irradiated with 365 nm UV, RNAs were cross-linked by AMT at the paired region, and survive DNase I, RNase T1 and RNase H treatments which digest DNA and single strand RNA. Cross-linked RNAs were ligated by T4 RNA ligase 1. After photoreversal of cross-linkages by 254 nm UV, the ligated RNAs could be sequenced and identified. b , bioinformatics analysis
    Figure Legend Snippet: Schematic overview of the modified protocol. a , wet experiment. Irradiated with 365 nm UV, RNAs were cross-linked by AMT at the paired region, and survive DNase I, RNase T1 and RNase H treatments which digest DNA and single strand RNA. Cross-linked RNAs were ligated by T4 RNA ligase 1. After photoreversal of cross-linkages by 254 nm UV, the ligated RNAs could be sequenced and identified. b , bioinformatics analysis

    Techniques Used: Modification, Irradiation

    4) Product Images from "Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine †"

    Article Title: Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine †

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1049

    ( A ) Schematic processing of the p1 16S rRNA. The extra-sequences of 115 nt and 33 nt, flanking the m 16S rRNA at its 5′ and 3′ ends, respectively, are shown on a grey background. RA and FA are the primers used for 3′5′ RACE analysis. The site of annealing of RA to m 16S rRNA, and that of FA to the reverse complement of the m 16S rRNA, are indicated by arrows. Figure not drawn to scale. ( B ) Expected sizes in bp of the RT-PCR products (amplicons) obtained from the different species of 16S rRNA ( p1 , p2 , p3 and m ) by 3′5′ RACE. ( C–F ) Agarose gel electrophoresis of RT-PCR products obtained by 3′5′ RACE from total RNA isolated from MC4100 bacteria grown at 30°C (C), or 44°C (D), or 45°C (E) or 46°C (F). Each RNA sample was thermo-denatured (lanes b), or not (lanes a) prior to the 3′5′ ligation. The sizes (in bp) of the molecular weight markers are indicated to the left of each gel (M). ( G ) The thermodenaturation step dissociates the complementary sequences present at the 3′ and 5′ends of the p1 16S rRNA, and therefore offers to all the 16S rRNA species an equal chance to access to the T4 RNA ligase.
    Figure Legend Snippet: ( A ) Schematic processing of the p1 16S rRNA. The extra-sequences of 115 nt and 33 nt, flanking the m 16S rRNA at its 5′ and 3′ ends, respectively, are shown on a grey background. RA and FA are the primers used for 3′5′ RACE analysis. The site of annealing of RA to m 16S rRNA, and that of FA to the reverse complement of the m 16S rRNA, are indicated by arrows. Figure not drawn to scale. ( B ) Expected sizes in bp of the RT-PCR products (amplicons) obtained from the different species of 16S rRNA ( p1 , p2 , p3 and m ) by 3′5′ RACE. ( C–F ) Agarose gel electrophoresis of RT-PCR products obtained by 3′5′ RACE from total RNA isolated from MC4100 bacteria grown at 30°C (C), or 44°C (D), or 45°C (E) or 46°C (F). Each RNA sample was thermo-denatured (lanes b), or not (lanes a) prior to the 3′5′ ligation. The sizes (in bp) of the molecular weight markers are indicated to the left of each gel (M). ( G ) The thermodenaturation step dissociates the complementary sequences present at the 3′ and 5′ends of the p1 16S rRNA, and therefore offers to all the 16S rRNA species an equal chance to access to the T4 RNA ligase.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Isolation, Ligation, Molecular Weight

    5) Product Images from "Integrated analysis of directly captured microRNA targets reveals the impact of microRNAs on mammalian transcriptome"

    Article Title: Integrated analysis of directly captured microRNA targets reveals the impact of microRNAs on mammalian transcriptome

    Journal: bioRxiv

    doi: 10.1101/672469

    Comparison of performance between CLEAR-CLIP captured and TargetScan predicted targets. a , Overlap between miR-200 CLEAR-CLIP genes with a 7mer or 8mer and TargetScan predictions for miR-200s. b , Log2 fold change in gene expression upon induction of the miR-200b cluster is shown for CLEAR-CLIP genes with a 7mer or 8mer and TargetScan predicted conserved sites as compared to genes without a miR-200 high confidence site and not predicted as conserved by TargetScan. c , Log2 fold change in gene expression upon induction of the miR-200b cluster is shown for genes only in CLEAR-CLIP, only predicted by TargetScan or in CLEAR-CLIP and TargetScan as compared to genes without a miR-200 high confidence site and not predicted by TargetScan. d , A portion of the Brd4 3’UTR is shown with miR-200 CLEAR-CLIP reads (top track), miR-200 seed sites (middle track) and TargetScan sites (bottom track) indicated. e , A portion of the Ammecr1l 3’UTR is shown with miR-200 CLEAR-CLIP reads (top track), miR-200 seed sites (middle track) and TargetScan sites (bottom track) indicated. f , A portion of the Tnrc6a 3’UTR is shown with miR-200 CLEAR-CLIP reads (top track) and miR-200 seed sites (bottom track) indicated. g , Overlap between miR-205 CLEAR-CLIP genes with a 7mer or 8mer and TargetScan predicted conserved sites for miR-205. h , Log2 fold change in gene expression upon induction of miR-205 is shown for CLEAR-CLIP genes with a 7mer or 8mer and TargetScan predicted sites as compared to genes without a miR-205 high confidence site and not predicted as conserved by TargetScan. i , Log2 fold change in gene expression upon induction of miR-205 is shown for genes only in CLEAR-CLIP, only predicted by TargetScan or in CLEAR-CLIP and TargetScan as compared to genes without a miR-205 high confidence site and not predicted by TargetScan. j , Overlap between all miRNA:mRNA CLEAR-CLIP interactions with a 7mer or 8mer and all conserved TargetScan predictions. For all CDF plots the number of genes is shown in parenthesis and p-values were calculated using the Kolmogorov–Smirnov test.
    Figure Legend Snippet: Comparison of performance between CLEAR-CLIP captured and TargetScan predicted targets. a , Overlap between miR-200 CLEAR-CLIP genes with a 7mer or 8mer and TargetScan predictions for miR-200s. b , Log2 fold change in gene expression upon induction of the miR-200b cluster is shown for CLEAR-CLIP genes with a 7mer or 8mer and TargetScan predicted conserved sites as compared to genes without a miR-200 high confidence site and not predicted as conserved by TargetScan. c , Log2 fold change in gene expression upon induction of the miR-200b cluster is shown for genes only in CLEAR-CLIP, only predicted by TargetScan or in CLEAR-CLIP and TargetScan as compared to genes without a miR-200 high confidence site and not predicted by TargetScan. d , A portion of the Brd4 3’UTR is shown with miR-200 CLEAR-CLIP reads (top track), miR-200 seed sites (middle track) and TargetScan sites (bottom track) indicated. e , A portion of the Ammecr1l 3’UTR is shown with miR-200 CLEAR-CLIP reads (top track), miR-200 seed sites (middle track) and TargetScan sites (bottom track) indicated. f , A portion of the Tnrc6a 3’UTR is shown with miR-200 CLEAR-CLIP reads (top track) and miR-200 seed sites (bottom track) indicated. g , Overlap between miR-205 CLEAR-CLIP genes with a 7mer or 8mer and TargetScan predicted conserved sites for miR-205. h , Log2 fold change in gene expression upon induction of miR-205 is shown for CLEAR-CLIP genes with a 7mer or 8mer and TargetScan predicted sites as compared to genes without a miR-205 high confidence site and not predicted as conserved by TargetScan. i , Log2 fold change in gene expression upon induction of miR-205 is shown for genes only in CLEAR-CLIP, only predicted by TargetScan or in CLEAR-CLIP and TargetScan as compared to genes without a miR-205 high confidence site and not predicted by TargetScan. j , Overlap between all miRNA:mRNA CLEAR-CLIP interactions with a 7mer or 8mer and all conserved TargetScan predictions. For all CDF plots the number of genes is shown in parenthesis and p-values were calculated using the Kolmogorov–Smirnov test.

    Techniques Used: Cross-linking Immunoprecipitation, Expressing

    6) Product Images from "Mapping the Human miRNA Interactome by CLASH Reveals Frequent Noncanonical Binding"

    Article Title: Mapping the Human miRNA Interactome by CLASH Reveals Frequent Noncanonical Binding

    Journal: Cell

    doi: 10.1016/j.cell.2013.03.043

    Overview of Experimental and Bioinformatic Procedures (A) Growing cells were UV irradiated, and PTH-AGO1 was purified. RNA fragmentation, ligation, cDNA synthesis, and sequencing of AGO1-associated RNAs allowed the identification of sites of AGO1 binding (as single reads) and RNA-RNA interactions at AGO1-binding sites (as chimeric reads). (B) Sequencing reads were mapped to a database of human transcripts using BLAST ( Altschul et al., 1990 ). Sequences reliably mapped to two different sites were folded in silico using UNAFold ( Markham and Zuker, 2008 ) to identify the interaction site of the RNA molecules that gave rise to the chimeric cDNA. (C) Example interaction between miR-196a/b and HOXC8 that was supported by chimeric reads (red), and a cluster of nonchimeric reads (green). The blue dashed line represents the location of the miRNA bit of chimera, and the red dashed line shows the 25 nt mRNA extension added during the analysis. The interaction was previously shown experimentally ( Li et al., 2010 ) and can be predicted by RNAhybrid ( Rehmsmeier et al., 2004 ). (D) Distribution of all miRNA interactions among various classes of RNAs. The main miRNA targets are mRNAs and are represented by 18,514 interactions. See also Figure S1 and Tables S1 and S2 A–S2C.
    Figure Legend Snippet: Overview of Experimental and Bioinformatic Procedures (A) Growing cells were UV irradiated, and PTH-AGO1 was purified. RNA fragmentation, ligation, cDNA synthesis, and sequencing of AGO1-associated RNAs allowed the identification of sites of AGO1 binding (as single reads) and RNA-RNA interactions at AGO1-binding sites (as chimeric reads). (B) Sequencing reads were mapped to a database of human transcripts using BLAST ( Altschul et al., 1990 ). Sequences reliably mapped to two different sites were folded in silico using UNAFold ( Markham and Zuker, 2008 ) to identify the interaction site of the RNA molecules that gave rise to the chimeric cDNA. (C) Example interaction between miR-196a/b and HOXC8 that was supported by chimeric reads (red), and a cluster of nonchimeric reads (green). The blue dashed line represents the location of the miRNA bit of chimera, and the red dashed line shows the 25 nt mRNA extension added during the analysis. The interaction was previously shown experimentally ( Li et al., 2010 ) and can be predicted by RNAhybrid ( Rehmsmeier et al., 2004 ). (D) Distribution of all miRNA interactions among various classes of RNAs. The main miRNA targets are mRNAs and are represented by 18,514 interactions. See also Figure S1 and Tables S1 and S2 A–S2C.

    Techniques Used: Irradiation, Purification, Ligation, Sequencing, Binding Assay, In Silico

    7) Product Images from "T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis"

    Article Title: T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-11-72

    Deadenylation activity of T4 RNA ligase 2 truncated mutants . 5'-adenylated DNA adapters were incubated with an excess of ligase (13.8 pmol), and 12.5% PEG 8000 at 16°C overnight. Oligonucleotide substrates are depicted schematically above the gel. The contents of each sample were resolved on denaturing 15% acrylamide gels and stained with SYBR Gold to visualize nucleic acid. Deadenylation of the DNA adapter (loss of 5'-App) is indicated by a band shift of ~1 nt towards the bottom of the gel. Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2 +MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP.
    Figure Legend Snippet: Deadenylation activity of T4 RNA ligase 2 truncated mutants . 5'-adenylated DNA adapters were incubated with an excess of ligase (13.8 pmol), and 12.5% PEG 8000 at 16°C overnight. Oligonucleotide substrates are depicted schematically above the gel. The contents of each sample were resolved on denaturing 15% acrylamide gels and stained with SYBR Gold to visualize nucleic acid. Deadenylation of the DNA adapter (loss of 5'-App) is indicated by a band shift of ~1 nt towards the bottom of the gel. Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2 +MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP.

    Techniques Used: Activity Assay, Incubation, Staining, Electrophoretic Mobility Shift Assay, Binding Assay

    Assaying the formation of side products by T4 RNA ligases . Intermolecular strand-joining reactions containing 5'-adenylated adapters, 21-mer 5'-PO 4 RNA acceptors, and ligase (1 pmol) were incubated at 16°C overnight in the presence of 12.5% PEG 8000. Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA. Products of the reaction were resolved on denaturing 15% acrylamide gels and stained with SYBR Gold. The bands corresponding to the input nucleic acids, the DNA adapter/RNA acceptor ligation product (39 bases), and larger side products are indicated. Ladder = size standard ladder, Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2 +MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP.
    Figure Legend Snippet: Assaying the formation of side products by T4 RNA ligases . Intermolecular strand-joining reactions containing 5'-adenylated adapters, 21-mer 5'-PO 4 RNA acceptors, and ligase (1 pmol) were incubated at 16°C overnight in the presence of 12.5% PEG 8000. Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA. Products of the reaction were resolved on denaturing 15% acrylamide gels and stained with SYBR Gold. The bands corresponding to the input nucleic acids, the DNA adapter/RNA acceptor ligation product (39 bases), and larger side products are indicated. Ladder = size standard ladder, Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2 +MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP.

    Techniques Used: Incubation, Staining, Ligation, Binding Assay

    Following AMP during ligation reactions with T4 RNA ligases . (A) 22-mer DNA adapters were 5'-adenylated with α- 32 P-labeled ATP (see materials and methods). Intermolecular strand-joining reactions containing 10 pmol radiolabeled DNA adapter, 5 pmol 21-mer 5'-PO 4 RNA acceptor, and ligase (1 pmol) were incubated overnight at 16°C in the presence of PEG 8000. Reaction products were resolved on a denaturing 15% acrylamide gel and radioactive molecules were visualized by exposure to Phosphor screens. The resulting products were either free AMP in solution (AMP*) or the adapter remaining adenylated (Ap*p-DNA). Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA. P* denotes 32 P-phosphate. (B) Determining the fate of AMP upon T4 RNA ligase-dependent deadenylation. Reactions containing radiolabeled DNA adapter (10 pmol) and ligase (14 pmol) were incubated overnight at 16°C in the presence of 12.5% PEG 8000. Oligonucleotide substrates are depicted schematically above the gel. P* denotes 32 P-phosphate. Reaction products were resolved and visualized as in (A). The resulting products were either free AMP in solution (AMP*), the adapter remaining adenylated (Ap*p-DNA), or AMP covalently bound to the ligase (AMP*-ligase). The lane labeled input contains only Ap*p-DNA. (C) Reactions identical to those in (B) were treated with Proteinase K prior to gel electrophoresis and detection. (D) Reactions containing 10 pmol radiolabeled DNA adapter, 5 pmol 28-mer [5'-PO 4 , 3'-blocked] RNA acceptor, and ligase (1 pmol) were incubated, resolved and detected as in (A). The resulting products were either free AMP in solution (AMP*), adenylated adapter (Ap*p-DNA), or Ap*p-28-mer RNA. The lane labeled RNA size control contains 5'- 32 PO 4 RNA, and the lane labeled input contains only Ap*p-DNA. Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA. P* denotes 32 P-phosphate. In all panels, Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2 +MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP.
    Figure Legend Snippet: Following AMP during ligation reactions with T4 RNA ligases . (A) 22-mer DNA adapters were 5'-adenylated with α- 32 P-labeled ATP (see materials and methods). Intermolecular strand-joining reactions containing 10 pmol radiolabeled DNA adapter, 5 pmol 21-mer 5'-PO 4 RNA acceptor, and ligase (1 pmol) were incubated overnight at 16°C in the presence of PEG 8000. Reaction products were resolved on a denaturing 15% acrylamide gel and radioactive molecules were visualized by exposure to Phosphor screens. The resulting products were either free AMP in solution (AMP*) or the adapter remaining adenylated (Ap*p-DNA). Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA. P* denotes 32 P-phosphate. (B) Determining the fate of AMP upon T4 RNA ligase-dependent deadenylation. Reactions containing radiolabeled DNA adapter (10 pmol) and ligase (14 pmol) were incubated overnight at 16°C in the presence of 12.5% PEG 8000. Oligonucleotide substrates are depicted schematically above the gel. P* denotes 32 P-phosphate. Reaction products were resolved and visualized as in (A). The resulting products were either free AMP in solution (AMP*), the adapter remaining adenylated (Ap*p-DNA), or AMP covalently bound to the ligase (AMP*-ligase). The lane labeled input contains only Ap*p-DNA. (C) Reactions identical to those in (B) were treated with Proteinase K prior to gel electrophoresis and detection. (D) Reactions containing 10 pmol radiolabeled DNA adapter, 5 pmol 28-mer [5'-PO 4 , 3'-blocked] RNA acceptor, and ligase (1 pmol) were incubated, resolved and detected as in (A). The resulting products were either free AMP in solution (AMP*), adenylated adapter (Ap*p-DNA), or Ap*p-28-mer RNA. The lane labeled RNA size control contains 5'- 32 PO 4 RNA, and the lane labeled input contains only Ap*p-DNA. Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA. P* denotes 32 P-phosphate. In all panels, Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2 +MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP.

    Techniques Used: Ligation, Labeling, Incubation, Acrylamide Gel Assay, Nucleic Acid Electrophoresis, Binding Assay

    Production of ligation side products by T4 RNA ligases . Intermolecular ligation reactions containing 5'-adenylated DNA adapters, 21-mer 5'-PO 4 RNA acceptors and ligase (1 pmol) were incubated at 16°C overnight with 12.5% PEG 8000. Products of the reactions were resolved on denaturing 15% acrylamide gels and stained with SYBR Gold. The bands corresponding to the input nucleic acids, the DNA adapter/RNA acceptor ligation product (39 bases), and larger side products are indicated. Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA.
    Figure Legend Snippet: Production of ligation side products by T4 RNA ligases . Intermolecular ligation reactions containing 5'-adenylated DNA adapters, 21-mer 5'-PO 4 RNA acceptors and ligase (1 pmol) were incubated at 16°C overnight with 12.5% PEG 8000. Products of the reactions were resolved on denaturing 15% acrylamide gels and stained with SYBR Gold. The bands corresponding to the input nucleic acids, the DNA adapter/RNA acceptor ligation product (39 bases), and larger side products are indicated. Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA.

    Techniques Used: Ligation, Incubation, Staining, Binding Assay

    Effect of pH on ligase intermolecular strand-joining activity . (A-D) Intermolecular strand-joining reactions were carried out with 10 pmol 5'-adenylated 17mer DNA, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, and ligase (1 pmol) for 1 hour at 25°C to assess the effect of pH on ligation efficiency. Ligation efficiency was determined by resolving the material in the reactions on denaturing 15% acrylamide gels and quantifying the amount of ligation product versus input nucleic acid. (E-H) Intermolecular strand-joining reactions were carried out with 10 pmol 5'-adenylated 17-mer DNA, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, and ligase (13.8 pmol) for 1 hour at 25°C to assess the effect of pH on ligation efficiency. Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP. Data are shown as the mean +/- SEM of at least three independent experiments.
    Figure Legend Snippet: Effect of pH on ligase intermolecular strand-joining activity . (A-D) Intermolecular strand-joining reactions were carried out with 10 pmol 5'-adenylated 17mer DNA, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, and ligase (1 pmol) for 1 hour at 25°C to assess the effect of pH on ligation efficiency. Ligation efficiency was determined by resolving the material in the reactions on denaturing 15% acrylamide gels and quantifying the amount of ligation product versus input nucleic acid. (E-H) Intermolecular strand-joining reactions were carried out with 10 pmol 5'-adenylated 17-mer DNA, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, and ligase (13.8 pmol) for 1 hour at 25°C to assess the effect of pH on ligation efficiency. Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP. Data are shown as the mean +/- SEM of at least three independent experiments.

    Techniques Used: Activity Assay, Labeling, Ligation, Binding Assay

    Analysis of intermolecular strand-joining over time . Strand-joining reactions were carried out with 10 pmol 5'-adenylated adapter, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, and ligase (1 pmol) over a span of 24 hours at 25°C to assess the progress of ligation reactions. Ligation efficiency was determined by resolving the material in the reactions on denaturing 15% acrylamide gels and quantifying the amount of ligation product versus input nucleic acid. Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP. Data are shown as the mean +/- SEM of at least three independent experiments.
    Figure Legend Snippet: Analysis of intermolecular strand-joining over time . Strand-joining reactions were carried out with 10 pmol 5'-adenylated adapter, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, and ligase (1 pmol) over a span of 24 hours at 25°C to assess the progress of ligation reactions. Ligation efficiency was determined by resolving the material in the reactions on denaturing 15% acrylamide gels and quantifying the amount of ligation product versus input nucleic acid. Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP. Data are shown as the mean +/- SEM of at least three independent experiments.

    Techniques Used: Labeling, Ligation, Binding Assay

    8) Product Images from "A Leaderless Genome Identified during Persistent Bovine Coronavirus Infection Is Associated with Attenuation of Gene Expression"

    Article Title: A Leaderless Genome Identified during Persistent Bovine Coronavirus Infection Is Associated with Attenuation of Gene Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0082176

    Identification of leaderless genomic RNA during BCoV persistent infection. (A) Strategy to identify positive-strand leaderless genomic RNA. Poly(A)-containing RNA was selected from total cellular RNA extracted from BCoV-persistently infected cells, treated with alkaline phosphatase, decapped with tobacco acid pyrophosphatase, head-to-tail ligated with T4 RNA ligase I, and used as the template for RT-PCR with the BCoV 5′ UTR-(+)-strand-specific primer 2: BCV107(+) (for RT) and BCoV 3′ UTR-(−)-strand-specific primer 1: BCV3′UTR1(−). (B) RT-PCR product synthesized by the method described in Fig. 1A. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) and with a size of less than 200 bp (lanes 6–7, marked with white arrowhead) were observed. (C) The upper panel shows part of the first 88-nt sequence of the 5′ UTR in the positive-strand BCoV genomic RNA. The positions (1 and 70) are given on the top of the sequence, and the intergenic sequence (IS) UCUAAAC is underlined. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of more than 200 bp from lane 7, as indicated with a black arrowhead in Fig. 1B. (D) The upper panel shows the sequence of the 5′UTR on the positive-strand BCoV genomic RNA, which lacks the first 69 nts; position 70 is given on the top of the sequence. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of less than 200 bp from lane 7, as indicated with a white arrowhead in Fig. 1B. (E) Control reactions to determine if the positive-strand leaderless genome is a degradation product. RT-PCR product was synthesized by the method described in Fig. 1A except RNA sample was not treated with alkaline phosphatase and tobacco acid pyrophosphatase. RT-PCR products with a length of more than 200 bp were detected. (F) Identification of negative-strand leaderless genomic RNA. Total cellular RNA was treated with tobacco acid pyrophosphatase and ligated with T4 RNA ligase I. RT-PCR product was synthesized by the method described in Fig. 1A except that primer BCV3′UTR1(−) was used for RT. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) were observed. M, ds DNA size markers in nt pairs. dpi: days postinfection.
    Figure Legend Snippet: Identification of leaderless genomic RNA during BCoV persistent infection. (A) Strategy to identify positive-strand leaderless genomic RNA. Poly(A)-containing RNA was selected from total cellular RNA extracted from BCoV-persistently infected cells, treated with alkaline phosphatase, decapped with tobacco acid pyrophosphatase, head-to-tail ligated with T4 RNA ligase I, and used as the template for RT-PCR with the BCoV 5′ UTR-(+)-strand-specific primer 2: BCV107(+) (for RT) and BCoV 3′ UTR-(−)-strand-specific primer 1: BCV3′UTR1(−). (B) RT-PCR product synthesized by the method described in Fig. 1A. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) and with a size of less than 200 bp (lanes 6–7, marked with white arrowhead) were observed. (C) The upper panel shows part of the first 88-nt sequence of the 5′ UTR in the positive-strand BCoV genomic RNA. The positions (1 and 70) are given on the top of the sequence, and the intergenic sequence (IS) UCUAAAC is underlined. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of more than 200 bp from lane 7, as indicated with a black arrowhead in Fig. 1B. (D) The upper panel shows the sequence of the 5′UTR on the positive-strand BCoV genomic RNA, which lacks the first 69 nts; position 70 is given on the top of the sequence. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of less than 200 bp from lane 7, as indicated with a white arrowhead in Fig. 1B. (E) Control reactions to determine if the positive-strand leaderless genome is a degradation product. RT-PCR product was synthesized by the method described in Fig. 1A except RNA sample was not treated with alkaline phosphatase and tobacco acid pyrophosphatase. RT-PCR products with a length of more than 200 bp were detected. (F) Identification of negative-strand leaderless genomic RNA. Total cellular RNA was treated with tobacco acid pyrophosphatase and ligated with T4 RNA ligase I. RT-PCR product was synthesized by the method described in Fig. 1A except that primer BCV3′UTR1(−) was used for RT. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) were observed. M, ds DNA size markers in nt pairs. dpi: days postinfection.

    Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction, Synthesized, Sequencing, Clone Assay

    9) Product Images from "A Leaderless Genome Identified during Persistent Bovine Coronavirus Infection Is Associated with Attenuation of Gene Expression"

    Article Title: A Leaderless Genome Identified during Persistent Bovine Coronavirus Infection Is Associated with Attenuation of Gene Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0082176

    Identification of leaderless genomic RNA during BCoV persistent infection. (A) Strategy to identify positive-strand leaderless genomic RNA. Poly(A)-containing RNA was selected from total cellular RNA extracted from BCoV-persistently infected cells, treated with alkaline phosphatase, decapped with tobacco acid pyrophosphatase, head-to-tail ligated with T4 RNA ligase I, and used as the template for RT-PCR with the BCoV 5′ UTR-(+)-strand-specific primer 2: BCV107(+) (for RT) and BCoV 3′ UTR-(−)-strand-specific primer 1: BCV3′UTR1(−). (B) RT-PCR product synthesized by the method described in Fig. 1A. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) and with a size of less than 200 bp (lanes 6–7, marked with white arrowhead) were observed. (C) The upper panel shows part of the first 88-nt sequence of the 5′ UTR in the positive-strand BCoV genomic RNA. The positions (1 and 70) are given on the top of the sequence, and the intergenic sequence (IS) UCUAAAC is underlined. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of more than 200 bp from lane 7, as indicated with a black arrowhead in Fig. 1B. (D) The upper panel shows the sequence of the 5′UTR on the positive-strand BCoV genomic RNA, which lacks the first 69 nts; position 70 is given on the top of the sequence. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of less than 200 bp from lane 7, as indicated with a white arrowhead in Fig. 1B. (E) Control reactions to determine if the positive-strand leaderless genome is a degradation product. RT-PCR product was synthesized by the method described in Fig. 1A except RNA sample was not treated with alkaline phosphatase and tobacco acid pyrophosphatase. RT-PCR products with a length of more than 200 bp were detected. (F) Identification of negative-strand leaderless genomic RNA. Total cellular RNA was treated with tobacco acid pyrophosphatase and ligated with T4 RNA ligase I. RT-PCR product was synthesized by the method described in Fig. 1A except that primer BCV3′UTR1(−) was used for RT. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) were observed. M, ds DNA size markers in nt pairs. dpi: days postinfection.
    Figure Legend Snippet: Identification of leaderless genomic RNA during BCoV persistent infection. (A) Strategy to identify positive-strand leaderless genomic RNA. Poly(A)-containing RNA was selected from total cellular RNA extracted from BCoV-persistently infected cells, treated with alkaline phosphatase, decapped with tobacco acid pyrophosphatase, head-to-tail ligated with T4 RNA ligase I, and used as the template for RT-PCR with the BCoV 5′ UTR-(+)-strand-specific primer 2: BCV107(+) (for RT) and BCoV 3′ UTR-(−)-strand-specific primer 1: BCV3′UTR1(−). (B) RT-PCR product synthesized by the method described in Fig. 1A. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) and with a size of less than 200 bp (lanes 6–7, marked with white arrowhead) were observed. (C) The upper panel shows part of the first 88-nt sequence of the 5′ UTR in the positive-strand BCoV genomic RNA. The positions (1 and 70) are given on the top of the sequence, and the intergenic sequence (IS) UCUAAAC is underlined. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of more than 200 bp from lane 7, as indicated with a black arrowhead in Fig. 1B. (D) The upper panel shows the sequence of the 5′UTR on the positive-strand BCoV genomic RNA, which lacks the first 69 nts; position 70 is given on the top of the sequence. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of less than 200 bp from lane 7, as indicated with a white arrowhead in Fig. 1B. (E) Control reactions to determine if the positive-strand leaderless genome is a degradation product. RT-PCR product was synthesized by the method described in Fig. 1A except RNA sample was not treated with alkaline phosphatase and tobacco acid pyrophosphatase. RT-PCR products with a length of more than 200 bp were detected. (F) Identification of negative-strand leaderless genomic RNA. Total cellular RNA was treated with tobacco acid pyrophosphatase and ligated with T4 RNA ligase I. RT-PCR product was synthesized by the method described in Fig. 1A except that primer BCV3′UTR1(−) was used for RT. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) were observed. M, ds DNA size markers in nt pairs. dpi: days postinfection.

    Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction, Synthesized, Sequencing, Clone Assay

    10) Product Images from "High-throughput determination of RNA structure by proximity ligation"

    Article Title: High-throughput determination of RNA structure by proximity ligation

    Journal: Nature biotechnology

    doi: 10.1038/nbt.3289

    RNA Proximity Ligation identifies structurally proximate regions within the complex secondary structures of S. cerevisiae ribosomal RNAs. a.) A schematic representation of the RPL method. Whole cells are spheroplasted with zymolyase and RNA is allowed to react with endogenous RNases. RNA ends are repaired in situ via T4 PNK to yield 5′-phosphate termini. Complexes are ligated overnight in the presence of T4 RNA Ligase I. Ligation products are cleaned up via acid guanidinium-phenol and subsequent DNase treatment, and subjected to Illumina TruSeq RNA-seq library preparation. These libraries are sequenced to map and count ligation junctions; b.-c.) We examined the distribution of ligation junctions as a function of distance from known base-pair partners in the 25S/5.8S rRNA and 18S rRNAs. Ligation products capture the structural proximity implied by base-pairing relationships, as evidenced by the enrichment for ligation junctions immediately near paired bases. Y-axes are shown as ligation counts per million reads analyzed. d.) Contact probability map for the eukaryotic 5.8S/25S rRNA based on RPL scores, which are calculated from the frequencies of ligation events between pairs of 21 nt windows ( Methods ). Lower inset : Ligation events, shown for bases 1300 to 1475 of the LSU rRNA in orange, primarily occur across digested single-stranded loops. RPL scores effectively smooth this noisy signal and are enriched for pairs of interacting regions. Plotted here are the 8,463 ligation events where both nucleotides fall within the displayed domain (compared to 17,029 ligation events where one nucleotide falls within the displayed domain and one does not, not shown). Right inset: RPL scores localize known pseudo-knots in the LSU rRNA structure, such as the interaction between bases 1727-1812 (shown in red) and bases 1941 – 2038 (shown in blue).
    Figure Legend Snippet: RNA Proximity Ligation identifies structurally proximate regions within the complex secondary structures of S. cerevisiae ribosomal RNAs. a.) A schematic representation of the RPL method. Whole cells are spheroplasted with zymolyase and RNA is allowed to react with endogenous RNases. RNA ends are repaired in situ via T4 PNK to yield 5′-phosphate termini. Complexes are ligated overnight in the presence of T4 RNA Ligase I. Ligation products are cleaned up via acid guanidinium-phenol and subsequent DNase treatment, and subjected to Illumina TruSeq RNA-seq library preparation. These libraries are sequenced to map and count ligation junctions; b.-c.) We examined the distribution of ligation junctions as a function of distance from known base-pair partners in the 25S/5.8S rRNA and 18S rRNAs. Ligation products capture the structural proximity implied by base-pairing relationships, as evidenced by the enrichment for ligation junctions immediately near paired bases. Y-axes are shown as ligation counts per million reads analyzed. d.) Contact probability map for the eukaryotic 5.8S/25S rRNA based on RPL scores, which are calculated from the frequencies of ligation events between pairs of 21 nt windows ( Methods ). Lower inset : Ligation events, shown for bases 1300 to 1475 of the LSU rRNA in orange, primarily occur across digested single-stranded loops. RPL scores effectively smooth this noisy signal and are enriched for pairs of interacting regions. Plotted here are the 8,463 ligation events where both nucleotides fall within the displayed domain (compared to 17,029 ligation events where one nucleotide falls within the displayed domain and one does not, not shown). Right inset: RPL scores localize known pseudo-knots in the LSU rRNA structure, such as the interaction between bases 1727-1812 (shown in red) and bases 1941 – 2038 (shown in blue).

    Techniques Used: Ligation, In Situ, RNA Sequencing Assay

    11) Product Images from "Detecting RNA-RNA interactions in E. coli using a modified CLASH method"

    Article Title: Detecting RNA-RNA interactions in E. coli using a modified CLASH method

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-3725-3

    Schematic overview of the modified protocol. a , wet experiment. Irradiated with 365 nm UV, RNAs were cross-linked by AMT at the paired region, and survive DNase I, RNase T1 and RNase H treatments which digest DNA and single strand RNA. Cross-linked RNAs were ligated by T4 RNA ligase 1. After photoreversal of cross-linkages by 254 nm UV, the ligated RNAs could be sequenced and identified. b , bioinformatics analysis
    Figure Legend Snippet: Schematic overview of the modified protocol. a , wet experiment. Irradiated with 365 nm UV, RNAs were cross-linked by AMT at the paired region, and survive DNase I, RNase T1 and RNase H treatments which digest DNA and single strand RNA. Cross-linked RNAs were ligated by T4 RNA ligase 1. After photoreversal of cross-linkages by 254 nm UV, the ligated RNAs could be sequenced and identified. b , bioinformatics analysis

    Techniques Used: Modification, Irradiation

    12) Product Images from "Tissue–selective effects of nucleolar stress and rDNA damage in developmental disorders"

    Article Title: Tissue–selective effects of nucleolar stress and rDNA damage in developmental disorders

    Journal: Nature

    doi: 10.1038/nature25449

    Inhibition of Pol I results in DNA damage in a subset of cells a , Representative immunofluorescence images of wild-type and TCOF1 +/− cNCCs stained with an antibody against γH2A.X; quantification is shown in b . c , Representative immunofluorescence images of DNA-damaged wild-type cNCCs stained with an antibody against γH2A.X after 1 h treatment with iPol I or actinomycin D (ActD); quantification is shown in d . e , Representative immunofluorescence images of DNA-damaged HeLa cells stained with an antibody against γH2A.X after 1 h treatment with iPol I; quantification is shown in f . For a – f , cells were collected from n = 3 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. ***P
    Figure Legend Snippet: Inhibition of Pol I results in DNA damage in a subset of cells a , Representative immunofluorescence images of wild-type and TCOF1 +/− cNCCs stained with an antibody against γH2A.X; quantification is shown in b . c , Representative immunofluorescence images of DNA-damaged wild-type cNCCs stained with an antibody against γH2A.X after 1 h treatment with iPol I or actinomycin D (ActD); quantification is shown in d . e , Representative immunofluorescence images of DNA-damaged HeLa cells stained with an antibody against γH2A.X after 1 h treatment with iPol I; quantification is shown in f . For a – f , cells were collected from n = 3 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. ***P

    Techniques Used: Inhibition, Immunofluorescence, Staining

    13) Product Images from "Mapping the Human miRNA Interactome by CLASH Reveals Frequent Noncanonical Binding"

    Article Title: Mapping the Human miRNA Interactome by CLASH Reveals Frequent Noncanonical Binding

    Journal: Cell

    doi: 10.1016/j.cell.2013.03.043

    Experimental Validation of CLASH Identified miR-92a Targets, Related to Figure 5 Changes in mRNA abundance upon miR-92a depletion in PTH-AGO1-HEK293 cells, measured by Affymetrix microarrays. The performance of various classes of miR-92a targets identified in CLASH analyses, and targets containing the miR-92a motif, are compared to transcripts containing a match to the miR-92a 7-mer seed sequence (positive control), to random transcripts, and to targets lacking a match to the miR-92a 7-mer seed (negative control). The left and right edge of the box represent 25th and 75th percentile, respectively. The ends of the whiskers show the minimum and maximum values of the data.
    Figure Legend Snippet: Experimental Validation of CLASH Identified miR-92a Targets, Related to Figure 5 Changes in mRNA abundance upon miR-92a depletion in PTH-AGO1-HEK293 cells, measured by Affymetrix microarrays. The performance of various classes of miR-92a targets identified in CLASH analyses, and targets containing the miR-92a motif, are compared to transcripts containing a match to the miR-92a 7-mer seed sequence (positive control), to random transcripts, and to targets lacking a match to the miR-92a 7-mer seed (negative control). The left and right edge of the box represent 25th and 75th percentile, respectively. The ends of the whiskers show the minimum and maximum values of the data.

    Techniques Used: Sequencing, Positive Control, Negative Control

    Overview of Experimental and Bioinformatic Procedures (A) Growing cells were UV irradiated, and PTH-AGO1 was purified. RNA fragmentation, ligation, cDNA synthesis, and sequencing of AGO1-associated RNAs allowed the identification of sites of AGO1 binding (as single reads) and RNA-RNA interactions at AGO1-binding sites (as chimeric reads). (B) Sequencing reads were mapped to a database of human transcripts using BLAST ( Altschul et al., 1990 ). Sequences reliably mapped to two different sites were folded in silico using UNAFold ( Markham and Zuker, 2008 ) to identify the interaction site of the RNA molecules that gave rise to the chimeric cDNA. (C) Example interaction between miR-196a/b and HOXC8 that was supported by chimeric reads (red), and a cluster of nonchimeric reads (green). The blue dashed line represents the location of the miRNA bit of chimera, and the red dashed line shows the 25 nt mRNA extension added during the analysis. The interaction was previously shown experimentally ( Li et al., 2010 ) and can be predicted by RNAhybrid ( Rehmsmeier et al., 2004 ). (D) Distribution of all miRNA interactions among various classes of RNAs. The main miRNA targets are mRNAs and are represented by 18,514 interactions. See also Figure S1 and Tables S1 and S2 A–S2C.
    Figure Legend Snippet: Overview of Experimental and Bioinformatic Procedures (A) Growing cells were UV irradiated, and PTH-AGO1 was purified. RNA fragmentation, ligation, cDNA synthesis, and sequencing of AGO1-associated RNAs allowed the identification of sites of AGO1 binding (as single reads) and RNA-RNA interactions at AGO1-binding sites (as chimeric reads). (B) Sequencing reads were mapped to a database of human transcripts using BLAST ( Altschul et al., 1990 ). Sequences reliably mapped to two different sites were folded in silico using UNAFold ( Markham and Zuker, 2008 ) to identify the interaction site of the RNA molecules that gave rise to the chimeric cDNA. (C) Example interaction between miR-196a/b and HOXC8 that was supported by chimeric reads (red), and a cluster of nonchimeric reads (green). The blue dashed line represents the location of the miRNA bit of chimera, and the red dashed line shows the 25 nt mRNA extension added during the analysis. The interaction was previously shown experimentally ( Li et al., 2010 ) and can be predicted by RNAhybrid ( Rehmsmeier et al., 2004 ). (D) Distribution of all miRNA interactions among various classes of RNAs. The main miRNA targets are mRNAs and are represented by 18,514 interactions. See also Figure S1 and Tables S1 and S2 A–S2C.

    Techniques Used: Irradiation, Purification, Ligation, Sequencing, Binding Assay, In Silico

    14) Product Images from "A Leaderless Genome Identified during Persistent Bovine Coronavirus Infection Is Associated with Attenuation of Gene Expression"

    Article Title: A Leaderless Genome Identified during Persistent Bovine Coronavirus Infection Is Associated with Attenuation of Gene Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0082176

    Identification of leaderless genomic RNA during BCoV persistent infection. (A) Strategy to identify positive-strand leaderless genomic RNA. Poly(A)-containing RNA was selected from total cellular RNA extracted from BCoV-persistently infected cells, treated with alkaline phosphatase, decapped with tobacco acid pyrophosphatase, head-to-tail ligated with T4 RNA ligase I, and used as the template for RT-PCR with the BCoV 5′ UTR-(+)-strand-specific primer 2: BCV107(+) (for RT) and BCoV 3′ UTR-(−)-strand-specific primer 1: BCV3′UTR1(−). (B) RT-PCR product synthesized by the method described in Fig. 1A. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) and with a size of less than 200 bp (lanes 6–7, marked with white arrowhead) were observed. (C) The upper panel shows part of the first 88-nt sequence of the 5′ UTR in the positive-strand BCoV genomic RNA. The positions (1 and 70) are given on the top of the sequence, and the intergenic sequence (IS) UCUAAAC is underlined. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of more than 200 bp from lane 7, as indicated with a black arrowhead in Fig. 1B. (D) The upper panel shows the sequence of the 5′UTR on the positive-strand BCoV genomic RNA, which lacks the first 69 nts; position 70 is given on the top of the sequence. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of less than 200 bp from lane 7, as indicated with a white arrowhead in Fig. 1B. (E) Control reactions to determine if the positive-strand leaderless genome is a degradation product. RT-PCR product was synthesized by the method described in Fig. 1A except RNA sample was not treated with alkaline phosphatase and tobacco acid pyrophosphatase. RT-PCR products with a length of more than 200 bp were detected. (F) Identification of negative-strand leaderless genomic RNA. Total cellular RNA was treated with tobacco acid pyrophosphatase and ligated with T4 RNA ligase I. RT-PCR product was synthesized by the method described in Fig. 1A except that primer BCV3′UTR1(−) was used for RT. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) were observed. M, ds DNA size markers in nt pairs. dpi: days postinfection.
    Figure Legend Snippet: Identification of leaderless genomic RNA during BCoV persistent infection. (A) Strategy to identify positive-strand leaderless genomic RNA. Poly(A)-containing RNA was selected from total cellular RNA extracted from BCoV-persistently infected cells, treated with alkaline phosphatase, decapped with tobacco acid pyrophosphatase, head-to-tail ligated with T4 RNA ligase I, and used as the template for RT-PCR with the BCoV 5′ UTR-(+)-strand-specific primer 2: BCV107(+) (for RT) and BCoV 3′ UTR-(−)-strand-specific primer 1: BCV3′UTR1(−). (B) RT-PCR product synthesized by the method described in Fig. 1A. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) and with a size of less than 200 bp (lanes 6–7, marked with white arrowhead) were observed. (C) The upper panel shows part of the first 88-nt sequence of the 5′ UTR in the positive-strand BCoV genomic RNA. The positions (1 and 70) are given on the top of the sequence, and the intergenic sequence (IS) UCUAAAC is underlined. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of more than 200 bp from lane 7, as indicated with a black arrowhead in Fig. 1B. (D) The upper panel shows the sequence of the 5′UTR on the positive-strand BCoV genomic RNA, which lacks the first 69 nts; position 70 is given on the top of the sequence. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of less than 200 bp from lane 7, as indicated with a white arrowhead in Fig. 1B. (E) Control reactions to determine if the positive-strand leaderless genome is a degradation product. RT-PCR product was synthesized by the method described in Fig. 1A except RNA sample was not treated with alkaline phosphatase and tobacco acid pyrophosphatase. RT-PCR products with a length of more than 200 bp were detected. (F) Identification of negative-strand leaderless genomic RNA. Total cellular RNA was treated with tobacco acid pyrophosphatase and ligated with T4 RNA ligase I. RT-PCR product was synthesized by the method described in Fig. 1A except that primer BCV3′UTR1(−) was used for RT. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) were observed. M, ds DNA size markers in nt pairs. dpi: days postinfection.

    Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction, Synthesized, Sequencing, Clone Assay

    15) Product Images from "Mapping the Human miRNA Interactome by CLASH Reveals Frequent Noncanonical Binding"

    Article Title: Mapping the Human miRNA Interactome by CLASH Reveals Frequent Noncanonical Binding

    Journal: Cell

    doi: 10.1016/j.cell.2013.03.043

    Experimental Validation of CLASH Identified miR-92a Targets, Related to Figure 5 Changes in mRNA abundance upon miR-92a depletion in PTH-AGO1-HEK293 cells, measured by Affymetrix microarrays. The performance of various classes of miR-92a targets identified in CLASH analyses, and targets containing the miR-92a motif, are compared to transcripts containing a match to the miR-92a 7-mer seed sequence (positive control), to random transcripts, and to targets lacking a match to the miR-92a 7-mer seed (negative control). The left and right edge of the box represent 25th and 75th percentile, respectively. The ends of the whiskers show the minimum and maximum values of the data.
    Figure Legend Snippet: Experimental Validation of CLASH Identified miR-92a Targets, Related to Figure 5 Changes in mRNA abundance upon miR-92a depletion in PTH-AGO1-HEK293 cells, measured by Affymetrix microarrays. The performance of various classes of miR-92a targets identified in CLASH analyses, and targets containing the miR-92a motif, are compared to transcripts containing a match to the miR-92a 7-mer seed sequence (positive control), to random transcripts, and to targets lacking a match to the miR-92a 7-mer seed (negative control). The left and right edge of the box represent 25th and 75th percentile, respectively. The ends of the whiskers show the minimum and maximum values of the data.

    Techniques Used: Sequencing, Positive Control, Negative Control

    Overview of Experimental and Bioinformatic Procedures (A) Growing cells were UV irradiated, and PTH-AGO1 was purified. RNA fragmentation, ligation, cDNA synthesis, and sequencing of AGO1-associated RNAs allowed the identification of sites of AGO1 binding (as single reads) and RNA-RNA interactions at AGO1-binding sites (as chimeric reads). (B) Sequencing reads were mapped to a database of human transcripts using BLAST ( Altschul et al., 1990 ). Sequences reliably mapped to two different sites were folded in silico using UNAFold ( Markham and Zuker, 2008 ) to identify the interaction site of the RNA molecules that gave rise to the chimeric cDNA. (C) Example interaction between miR-196a/b and HOXC8 that was supported by chimeric reads (red), and a cluster of nonchimeric reads (green). The blue dashed line represents the location of the miRNA bit of chimera, and the red dashed line shows the 25 nt mRNA extension added during the analysis. The interaction was previously shown experimentally ( Li et al., 2010 ) and can be predicted by RNAhybrid ( Rehmsmeier et al., 2004 ). (D) Distribution of all miRNA interactions among various classes of RNAs. The main miRNA targets are mRNAs and are represented by 18,514 interactions. See also Figure S1 and Tables S1 and S2 A–S2C.
    Figure Legend Snippet: Overview of Experimental and Bioinformatic Procedures (A) Growing cells were UV irradiated, and PTH-AGO1 was purified. RNA fragmentation, ligation, cDNA synthesis, and sequencing of AGO1-associated RNAs allowed the identification of sites of AGO1 binding (as single reads) and RNA-RNA interactions at AGO1-binding sites (as chimeric reads). (B) Sequencing reads were mapped to a database of human transcripts using BLAST ( Altschul et al., 1990 ). Sequences reliably mapped to two different sites were folded in silico using UNAFold ( Markham and Zuker, 2008 ) to identify the interaction site of the RNA molecules that gave rise to the chimeric cDNA. (C) Example interaction between miR-196a/b and HOXC8 that was supported by chimeric reads (red), and a cluster of nonchimeric reads (green). The blue dashed line represents the location of the miRNA bit of chimera, and the red dashed line shows the 25 nt mRNA extension added during the analysis. The interaction was previously shown experimentally ( Li et al., 2010 ) and can be predicted by RNAhybrid ( Rehmsmeier et al., 2004 ). (D) Distribution of all miRNA interactions among various classes of RNAs. The main miRNA targets are mRNAs and are represented by 18,514 interactions. See also Figure S1 and Tables S1 and S2 A–S2C.

    Techniques Used: Irradiation, Purification, Ligation, Sequencing, Binding Assay, In Silico

    16) Product Images from "Blocking of targeted microRNAs from next-generation sequencing libraries"

    Article Title: Blocking of targeted microRNAs from next-generation sequencing libraries

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv724

    Modification of miRNA sequencing library generation protocol to allow for blocking of targeted species. ( A ) In the standard protocol, a pre-adenylated adaptor is ligated to the 3′ end of a small RNA pool using T4 RNA Ligase 2, truncated. Subsequently, a second adaptor is added to the 5′ end of the miRNA with T4 RNA Ligase 1, followed by reverse transcription and PCR. ( B ) In our modification, a hairpin oligonucleotide with an overhang complementary to the 5′ end of the targeted miRNA is attached via ligation with T4 DNA Ligase to the 5′ end of the miRNA subsequent to the ligation of the adaptor to the 3′ end. This prevents the ligation of the second adaptor to the 5′ end of the miRNA, resulting in a product that does not amplify during PCR. ( C ) Sequencing libraries were generated from human heart total RNA using a titration of a blocking oligonucleotide targeting hsa-miR-16–5p. The fraction of hsa-miR-16–5p present in the blocked library compared to the unblocked library is shown on the y-axis.
    Figure Legend Snippet: Modification of miRNA sequencing library generation protocol to allow for blocking of targeted species. ( A ) In the standard protocol, a pre-adenylated adaptor is ligated to the 3′ end of a small RNA pool using T4 RNA Ligase 2, truncated. Subsequently, a second adaptor is added to the 5′ end of the miRNA with T4 RNA Ligase 1, followed by reverse transcription and PCR. ( B ) In our modification, a hairpin oligonucleotide with an overhang complementary to the 5′ end of the targeted miRNA is attached via ligation with T4 DNA Ligase to the 5′ end of the miRNA subsequent to the ligation of the adaptor to the 3′ end. This prevents the ligation of the second adaptor to the 5′ end of the miRNA, resulting in a product that does not amplify during PCR. ( C ) Sequencing libraries were generated from human heart total RNA using a titration of a blocking oligonucleotide targeting hsa-miR-16–5p. The fraction of hsa-miR-16–5p present in the blocked library compared to the unblocked library is shown on the y-axis.

    Techniques Used: Modification, Sequencing, Blocking Assay, Polymerase Chain Reaction, Ligation, Generated, Titration

    17) Product Images from "A Leaderless Genome Identified during Persistent Bovine Coronavirus Infection Is Associated with Attenuation of Gene Expression"

    Article Title: A Leaderless Genome Identified during Persistent Bovine Coronavirus Infection Is Associated with Attenuation of Gene Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0082176

    Identification of leaderless genomic RNA during BCoV persistent infection. (A) Strategy to identify positive-strand leaderless genomic RNA. Poly(A)-containing RNA was selected from total cellular RNA extracted from BCoV-persistently infected cells, treated with alkaline phosphatase, decapped with tobacco acid pyrophosphatase, head-to-tail ligated with T4 RNA ligase I, and used as the template for RT-PCR with the BCoV 5′ UTR-(+)-strand-specific primer 2: BCV107(+) (for RT) and BCoV 3′ UTR-(−)-strand-specific primer 1: BCV3′UTR1(−). (B) RT-PCR product synthesized by the method described in Fig. 1A. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) and with a size of less than 200 bp (lanes 6–7, marked with white arrowhead) were observed. (C) The upper panel shows part of the first 88-nt sequence of the 5′ UTR in the positive-strand BCoV genomic RNA. The positions (1 and 70) are given on the top of the sequence, and the intergenic sequence (IS) UCUAAAC is underlined. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of more than 200 bp from lane 7, as indicated with a black arrowhead in Fig. 1B. (D) The upper panel shows the sequence of the 5′UTR on the positive-strand BCoV genomic RNA, which lacks the first 69 nts; position 70 is given on the top of the sequence. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of less than 200 bp from lane 7, as indicated with a white arrowhead in Fig. 1B. (E) Control reactions to determine if the positive-strand leaderless genome is a degradation product. RT-PCR product was synthesized by the method described in Fig. 1A except RNA sample was not treated with alkaline phosphatase and tobacco acid pyrophosphatase. RT-PCR products with a length of more than 200 bp were detected. (F) Identification of negative-strand leaderless genomic RNA. Total cellular RNA was treated with tobacco acid pyrophosphatase and ligated with T4 RNA ligase I. RT-PCR product was synthesized by the method described in Fig. 1A except that primer BCV3′UTR1(−) was used for RT. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) were observed. M, ds DNA size markers in nt pairs. dpi: days postinfection.
    Figure Legend Snippet: Identification of leaderless genomic RNA during BCoV persistent infection. (A) Strategy to identify positive-strand leaderless genomic RNA. Poly(A)-containing RNA was selected from total cellular RNA extracted from BCoV-persistently infected cells, treated with alkaline phosphatase, decapped with tobacco acid pyrophosphatase, head-to-tail ligated with T4 RNA ligase I, and used as the template for RT-PCR with the BCoV 5′ UTR-(+)-strand-specific primer 2: BCV107(+) (for RT) and BCoV 3′ UTR-(−)-strand-specific primer 1: BCV3′UTR1(−). (B) RT-PCR product synthesized by the method described in Fig. 1A. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) and with a size of less than 200 bp (lanes 6–7, marked with white arrowhead) were observed. (C) The upper panel shows part of the first 88-nt sequence of the 5′ UTR in the positive-strand BCoV genomic RNA. The positions (1 and 70) are given on the top of the sequence, and the intergenic sequence (IS) UCUAAAC is underlined. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of more than 200 bp from lane 7, as indicated with a black arrowhead in Fig. 1B. (D) The upper panel shows the sequence of the 5′UTR on the positive-strand BCoV genomic RNA, which lacks the first 69 nts; position 70 is given on the top of the sequence. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of less than 200 bp from lane 7, as indicated with a white arrowhead in Fig. 1B. (E) Control reactions to determine if the positive-strand leaderless genome is a degradation product. RT-PCR product was synthesized by the method described in Fig. 1A except RNA sample was not treated with alkaline phosphatase and tobacco acid pyrophosphatase. RT-PCR products with a length of more than 200 bp were detected. (F) Identification of negative-strand leaderless genomic RNA. Total cellular RNA was treated with tobacco acid pyrophosphatase and ligated with T4 RNA ligase I. RT-PCR product was synthesized by the method described in Fig. 1A except that primer BCV3′UTR1(−) was used for RT. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) were observed. M, ds DNA size markers in nt pairs. dpi: days postinfection.

    Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction, Synthesized, Sequencing, Clone Assay

    18) Product Images from "Mapping interactions between the RNA chaperone FinO and its RNA targets"

    Article Title: Mapping interactions between the RNA chaperone FinO and its RNA targets

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr025

    FinO binding to SLII requires a terminal 3′-OH on the 3′-tail of SLII. Native gels (8%) of binding reactions between FinO constructs and SLII RNA derivatives. ( A ) FinO does not bind SLII RNA containing a 3′,5′-cytidine diphosphate 3′-terminus. T4 RNA ligase I was used to ligate 3′,5′-cytidine [5′- 32 P] disphosphate (pCp) to the 3′-tail resulting in a 3′-phosphate. NP, no protein. Triangles represent increasing concentrations of FinO or the indicated mutants: 0.25, 0.5, 1, 2.5, 5 and 10 µM. The positions of free 32 P-SLII and the FinO- 32 P-SLII are noted by arrows. ( B ) Treatment of SLII RNA containing a 3′,5′-cytidine diphosphate 3′-terminus to give a 2′,3′ cis -diol (3′-hydroxyl) restores FinO binding. ( C ) Oxidation of SLII with sodium periodate to give a 2′,3′ dialdehyde reduces binding affinity. The protein concentrations were 1 µM in each of the binding reactions in B and C.
    Figure Legend Snippet: FinO binding to SLII requires a terminal 3′-OH on the 3′-tail of SLII. Native gels (8%) of binding reactions between FinO constructs and SLII RNA derivatives. ( A ) FinO does not bind SLII RNA containing a 3′,5′-cytidine diphosphate 3′-terminus. T4 RNA ligase I was used to ligate 3′,5′-cytidine [5′- 32 P] disphosphate (pCp) to the 3′-tail resulting in a 3′-phosphate. NP, no protein. Triangles represent increasing concentrations of FinO or the indicated mutants: 0.25, 0.5, 1, 2.5, 5 and 10 µM. The positions of free 32 P-SLII and the FinO- 32 P-SLII are noted by arrows. ( B ) Treatment of SLII RNA containing a 3′,5′-cytidine diphosphate 3′-terminus to give a 2′,3′ cis -diol (3′-hydroxyl) restores FinO binding. ( C ) Oxidation of SLII with sodium periodate to give a 2′,3′ dialdehyde reduces binding affinity. The protein concentrations were 1 µM in each of the binding reactions in B and C.

    Techniques Used: Binding Assay, Construct

    19) Product Images from "Mapping interactions between the RNA chaperone FinO and its RNA targets"

    Article Title: Mapping interactions between the RNA chaperone FinO and its RNA targets

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr025

    FinO binding to SLII requires a terminal 3′-OH on the 3′-tail of SLII. Native gels (8%) of binding reactions between FinO constructs and SLII RNA derivatives. ( A ) FinO does not bind SLII RNA containing a 3′,5′-cytidine diphosphate 3′-terminus. T4 RNA ligase I was used to ligate 3′,5′-cytidine [5′- 32 P] disphosphate (pCp) to the 3′-tail resulting in a 3′-phosphate. NP, no protein. Triangles represent increasing concentrations of FinO or the indicated mutants: 0.25, 0.5, 1, 2.5, 5 and 10 µM. The positions of free 32 P-SLII and the FinO- 32 P-SLII are noted by arrows. ( B ) Treatment of SLII RNA containing a 3′,5′-cytidine diphosphate 3′-terminus to give a 2′,3′ cis -diol (3′-hydroxyl) restores FinO binding. ( C ) Oxidation of SLII with sodium periodate to give a 2′,3′ dialdehyde reduces binding affinity. The protein concentrations were 1 µM in each of the binding reactions in B and C.
    Figure Legend Snippet: FinO binding to SLII requires a terminal 3′-OH on the 3′-tail of SLII. Native gels (8%) of binding reactions between FinO constructs and SLII RNA derivatives. ( A ) FinO does not bind SLII RNA containing a 3′,5′-cytidine diphosphate 3′-terminus. T4 RNA ligase I was used to ligate 3′,5′-cytidine [5′- 32 P] disphosphate (pCp) to the 3′-tail resulting in a 3′-phosphate. NP, no protein. Triangles represent increasing concentrations of FinO or the indicated mutants: 0.25, 0.5, 1, 2.5, 5 and 10 µM. The positions of free 32 P-SLII and the FinO- 32 P-SLII are noted by arrows. ( B ) Treatment of SLII RNA containing a 3′,5′-cytidine diphosphate 3′-terminus to give a 2′,3′ cis -diol (3′-hydroxyl) restores FinO binding. ( C ) Oxidation of SLII with sodium periodate to give a 2′,3′ dialdehyde reduces binding affinity. The protein concentrations were 1 µM in each of the binding reactions in B and C.

    Techniques Used: Binding Assay, Construct

    20) Product Images from "3′ Branch ligation: a novel method to ligate non-complementary DNA to recessed or internal 3′OH ends in DNA or RNA"

    Article Title: 3′ Branch ligation: a novel method to ligate non-complementary DNA to recessed or internal 3′OH ends in DNA or RNA

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    doi: 10.1093/dnares/dsy037

    3′ Branch ligation at the 3′ end of RNA in DNA/RNA hybrid. Schematic representation of 3′-branch ligation on a DNA/RNA hybrid with a 20‐bp complimentary region. We tested whether blunt-end DNA donors would ligate to the 3 ′ -recessive end of DNA and/or to the 3 ′ -recessive end of RNA. DNA(ON-21) hybridizes with the RNA strand (a), whereas DNA(ON-23) cannot hybridize with the RNA strand (b). (c, d) Gel analysis of size shift of ligated products using 6% denaturing polyacrylamide gel. The red arrowheads correspond to the RNA substrate (29 nt), and the green arrowhead corresponds to DNA substrate (80 nt). The purple arrowhead corresponds to donor-ligated RNA substrates. If ligation occurs, the substrate size would shift up by 20 nt. (c) Lanes 1 and 2, experimental duplicates; lanes 7–10, no-ligase controls; 10% PEG was added with T4 DNA ligase. (d) Lane 1, no-ligase control; lanes 2, 3, and 8, T4 DNA ligase with 10% PEG; lanes 4, 5, and 9, T4 RNA ligase 1 with 20% DMSO; lanes 6, 7, and 10, T4 RNA ligase 2 with 20% DMSO.
    Figure Legend Snippet: 3′ Branch ligation at the 3′ end of RNA in DNA/RNA hybrid. Schematic representation of 3′-branch ligation on a DNA/RNA hybrid with a 20‐bp complimentary region. We tested whether blunt-end DNA donors would ligate to the 3 ′ -recessive end of DNA and/or to the 3 ′ -recessive end of RNA. DNA(ON-21) hybridizes with the RNA strand (a), whereas DNA(ON-23) cannot hybridize with the RNA strand (b). (c, d) Gel analysis of size shift of ligated products using 6% denaturing polyacrylamide gel. The red arrowheads correspond to the RNA substrate (29 nt), and the green arrowhead corresponds to DNA substrate (80 nt). The purple arrowhead corresponds to donor-ligated RNA substrates. If ligation occurs, the substrate size would shift up by 20 nt. (c) Lanes 1 and 2, experimental duplicates; lanes 7–10, no-ligase controls; 10% PEG was added with T4 DNA ligase. (d) Lane 1, no-ligase control; lanes 2, 3, and 8, T4 DNA ligase with 10% PEG; lanes 4, 5, and 9, T4 RNA ligase 1 with 20% DMSO; lanes 6, 7, and 10, T4 RNA ligase 2 with 20% DMSO.

    Techniques Used: Ligation

    21) Product Images from "Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine †"

    Article Title: Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine †

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1049

    ( A ) Schematic processing of the p1 16S rRNA. The extra-sequences of 115 nt and 33 nt, flanking the m 16S rRNA at its 5′ and 3′ ends, respectively, are shown on a grey background. RA and FA are the primers used for 3′5′ RACE analysis. The site of annealing of RA to m 16S rRNA, and that of FA to the reverse complement of the m 16S rRNA, are indicated by arrows. Figure not drawn to scale. ( B ) Expected sizes in bp of the RT-PCR products (amplicons) obtained from the different species of 16S rRNA ( p1 , p2 , p3 and m ) by 3′5′ RACE. ( C–F ) Agarose gel electrophoresis of RT-PCR products obtained by 3′5′ RACE from total RNA isolated from MC4100 bacteria grown at 30°C (C), or 44°C (D), or 45°C (E) or 46°C (F). Each RNA sample was thermo-denatured (lanes b), or not (lanes a) prior to the 3′5′ ligation. The sizes (in bp) of the molecular weight markers are indicated to the left of each gel (M). ( G ) The thermodenaturation step dissociates the complementary sequences present at the 3′ and 5′ends of the p1 16S rRNA, and therefore offers to all the 16S rRNA species an equal chance to access to the T4 RNA ligase.
    Figure Legend Snippet: ( A ) Schematic processing of the p1 16S rRNA. The extra-sequences of 115 nt and 33 nt, flanking the m 16S rRNA at its 5′ and 3′ ends, respectively, are shown on a grey background. RA and FA are the primers used for 3′5′ RACE analysis. The site of annealing of RA to m 16S rRNA, and that of FA to the reverse complement of the m 16S rRNA, are indicated by arrows. Figure not drawn to scale. ( B ) Expected sizes in bp of the RT-PCR products (amplicons) obtained from the different species of 16S rRNA ( p1 , p2 , p3 and m ) by 3′5′ RACE. ( C–F ) Agarose gel electrophoresis of RT-PCR products obtained by 3′5′ RACE from total RNA isolated from MC4100 bacteria grown at 30°C (C), or 44°C (D), or 45°C (E) or 46°C (F). Each RNA sample was thermo-denatured (lanes b), or not (lanes a) prior to the 3′5′ ligation. The sizes (in bp) of the molecular weight markers are indicated to the left of each gel (M). ( G ) The thermodenaturation step dissociates the complementary sequences present at the 3′ and 5′ends of the p1 16S rRNA, and therefore offers to all the 16S rRNA species an equal chance to access to the T4 RNA ligase.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Isolation, Ligation, Molecular Weight

    22) Product Images from "Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation"

    Article Title: Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0167009

    Optimization of the 3´ adapter ligation step. Synthetic Let-7d-5p (NNN) miRNA was ligated to the 3´ adapter using the same ligation conditions as the CleanTag library prep workflow step 1. A) Yield increase with addition of PEG 8000 using T4 RNA Ligase 2, truncated KQ and modified 3´ adapter (MP (n-1)). B) Specificity comparison between ligases used in 3´ ligation step: 1) T4 RNA Ligase 2, truncated; 2) T4 RNA Ligase 2, truncated KQ; 3) T4 RNA Ligase 1; 4) No Ligase. Both unmodified and modified (MP (n-1)) 3´ adapters were tested. Side products indicated with red arrows.
    Figure Legend Snippet: Optimization of the 3´ adapter ligation step. Synthetic Let-7d-5p (NNN) miRNA was ligated to the 3´ adapter using the same ligation conditions as the CleanTag library prep workflow step 1. A) Yield increase with addition of PEG 8000 using T4 RNA Ligase 2, truncated KQ and modified 3´ adapter (MP (n-1)). B) Specificity comparison between ligases used in 3´ ligation step: 1) T4 RNA Ligase 2, truncated; 2) T4 RNA Ligase 2, truncated KQ; 3) T4 RNA Ligase 1; 4) No Ligase. Both unmodified and modified (MP (n-1)) 3´ adapters were tested. Side products indicated with red arrows.

    Techniques Used: Ligation, Modification

    Ligation screen for modified adapters that suppress adapter dimer formation. Example of modifications screened on the 5´adapter for ligation suppression against the Luo 3΄ Adapter with MP (n-1). Unmodified adapters were shown for comparison (U = unmodified). Adapter concentrations were 1 μM. Ligations performed with 10 U T4 RNA Ligase 1, 1 mM ATP, and 20% PEG, incubated for 2 hours at 37°C. Candidate modifications which reduce dimer formation are highlighted with blue box.
    Figure Legend Snippet: Ligation screen for modified adapters that suppress adapter dimer formation. Example of modifications screened on the 5´adapter for ligation suppression against the Luo 3΄ Adapter with MP (n-1). Unmodified adapters were shown for comparison (U = unmodified). Adapter concentrations were 1 μM. Ligations performed with 10 U T4 RNA Ligase 1, 1 mM ATP, and 20% PEG, incubated for 2 hours at 37°C. Candidate modifications which reduce dimer formation are highlighted with blue box.

    Techniques Used: Ligation, Modification, Incubation

    23) Product Images from "Capture and sequence analysis of RNAs with terminal 2\u2032,3\u2032-cyclic phosphates"

    Article Title: Capture and sequence analysis of RNAs with terminal 2\u2032,3\u2032-cyclic phosphates

    Journal: RNA

    doi: 10.1261/rna.1934910

    RNA termini and tRNA splicing. ( A ) RNA termini with a 2′,3′- cis diol ( top ; T4 RNA ligase 1 substrate) or 2′,3′-cylic phosphate ( bottom ; A. thaliana tRNA ligase substrate). ( B ) The tRNA splicing pathway generates intermediates
    Figure Legend Snippet: RNA termini and tRNA splicing. ( A ) RNA termini with a 2′,3′- cis diol ( top ; T4 RNA ligase 1 substrate) or 2′,3′-cylic phosphate ( bottom ; A. thaliana tRNA ligase substrate). ( B ) The tRNA splicing pathway generates intermediates

    Techniques Used:

    24) Product Images from "Blocking of targeted microRNAs from next-generation sequencing libraries"

    Article Title: Blocking of targeted microRNAs from next-generation sequencing libraries

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv724

    Modification of miRNA sequencing library generation protocol to allow for blocking of targeted species. ( A ) In the standard protocol, a pre-adenylated adaptor is ligated to the 3′ end of a small RNA pool using T4 RNA Ligase 2, truncated. Subsequently, a second adaptor is added to the 5′ end of the miRNA with T4 RNA Ligase 1, followed by reverse transcription and PCR. ( B ) In our modification, a hairpin oligonucleotide with an overhang complementary to the 5′ end of the targeted miRNA is attached via ligation with T4 DNA Ligase to the 5′ end of the miRNA subsequent to the ligation of the adaptor to the 3′ end. This prevents the ligation of the second adaptor to the 5′ end of the miRNA, resulting in a product that does not amplify during PCR. ( C ) Sequencing libraries were generated from human heart total RNA using a titration of a blocking oligonucleotide targeting hsa-miR-16–5p. The fraction of hsa-miR-16–5p present in the blocked library compared to the unblocked library is shown on the y-axis.
    Figure Legend Snippet: Modification of miRNA sequencing library generation protocol to allow for blocking of targeted species. ( A ) In the standard protocol, a pre-adenylated adaptor is ligated to the 3′ end of a small RNA pool using T4 RNA Ligase 2, truncated. Subsequently, a second adaptor is added to the 5′ end of the miRNA with T4 RNA Ligase 1, followed by reverse transcription and PCR. ( B ) In our modification, a hairpin oligonucleotide with an overhang complementary to the 5′ end of the targeted miRNA is attached via ligation with T4 DNA Ligase to the 5′ end of the miRNA subsequent to the ligation of the adaptor to the 3′ end. This prevents the ligation of the second adaptor to the 5′ end of the miRNA, resulting in a product that does not amplify during PCR. ( C ) Sequencing libraries were generated from human heart total RNA using a titration of a blocking oligonucleotide targeting hsa-miR-16–5p. The fraction of hsa-miR-16–5p present in the blocked library compared to the unblocked library is shown on the y-axis.

    Techniques Used: Modification, Sequencing, Blocking Assay, Polymerase Chain Reaction, Ligation, Generated, Titration

    25) Product Images from "Capture and sequence analysis of RNAs with terminal 2\u2032,3\u2032-cyclic phosphates"

    Article Title: Capture and sequence analysis of RNAs with terminal 2\u2032,3\u2032-cyclic phosphates

    Journal: RNA

    doi: 10.1261/rna.1934910

    RNA termini and tRNA splicing. ( A ) RNA termini with a 2′,3′- cis diol ( top ; T4 RNA ligase 1 substrate) or 2′,3′-cylic phosphate ( bottom ; A. thaliana tRNA ligase substrate). ( B ) The tRNA splicing pathway generates intermediates
    Figure Legend Snippet: RNA termini and tRNA splicing. ( A ) RNA termini with a 2′,3′- cis diol ( top ; T4 RNA ligase 1 substrate) or 2′,3′-cylic phosphate ( bottom ; A. thaliana tRNA ligase substrate). ( B ) The tRNA splicing pathway generates intermediates

    Techniques Used:

    Related Articles

    Sequencing:

    Article Title: A Leaderless Genome Identified during Persistent Bovine Coronavirus Infection Is Associated with Attenuation of Gene Expression
    Article Snippet: .. To determine the terminal sequence of viral negative-strand genomic RNA and sgmRNA, total cellular RNA was treated with tobacco acid pyrophosphatase (Epicentre), ligated with T4 RNA ligase I (New England Biolabs) and primer 1: BCV3′UTR1(−) was used for RT; for PCR, primers BCV3′UTR(−) and BCV107(+), and primers BCV3′UTR(−) and RYN(+) were used for determining terminal sequence of negative-strand genomic RNA and subgenomic mRNA, respectively. .. The resulting 50-µl PCR mixture was heated to 94°C for 2 min and subjected to 50 cycles of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C.

    Polymerase Chain Reaction:

    Article Title: A Leaderless Genome Identified during Persistent Bovine Coronavirus Infection Is Associated with Attenuation of Gene Expression
    Article Snippet: .. To determine the terminal sequence of viral negative-strand genomic RNA and sgmRNA, total cellular RNA was treated with tobacco acid pyrophosphatase (Epicentre), ligated with T4 RNA ligase I (New England Biolabs) and primer 1: BCV3′UTR1(−) was used for RT; for PCR, primers BCV3′UTR(−) and BCV107(+), and primers BCV3′UTR(−) and RYN(+) were used for determining terminal sequence of negative-strand genomic RNA and subgenomic mRNA, respectively. .. The resulting 50-µl PCR mixture was heated to 94°C for 2 min and subjected to 50 cycles of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C.

    Incubation:

    Article Title: A Leaderless Genome Identified during Persistent Bovine Coronavirus Infection Is Associated with Attenuation of Gene Expression
    Article Snippet: .. A 3-µl aliquot of 10X ligase buffer and 2 U (in 2 µl) of T4 RNA ligase I (New England Biolabs) were added, and the mixture was incubated for 16 h at 16°C. .. After ligation, RNA was phenol-chloroform-extracted and quantitated, and 1 µg of ligated RNA was used for an RT reaction to synthesize cDNA with SuperScript III reverse transcriptase (Invitrogen).

    Article Title: A Leaderless Genome Identified during Persistent Bovine Coronavirus Infection Is Associated with Attenuation of Gene Expression
    Article Snippet: .. The extracted RNA in 25 µl of water, 3 µl of 10X ligase buffer, and 2 U (in 2 µl) of T4 RNA ligase I (New England Biolabs) were combined, and the mixture was incubated for 16 h at 16°C. ..

    Article Title: High-throughput determination of RNA structure by proximity ligation
    Article Snippet: .. Following end-repair, complexes were immediately transferred to 450 uL ligation reaction mix (50 uL 10X T4 DNA Ligase Buffer w/ 10 mM ATP (NEB); 5 uL SuperASE-In (Ambion), 12.5 uL T4 RNA Ligase I (NEB), 382.5 uL 1X PBS w/ 0.2% IGEPAL), and incubated overnight in a 16 °C water bath, after which complexes were added to 1.5 mL TriZOL (Ambion). .. Samples were then purified using Direct-ZOL spin columns (Zymo) according to manufacturer's protocols.

    other:

    Article Title: Detecting RNA-RNA interactions in E. coli using a modified CLASH method
    Article Snippet: Free RNA overhangs adjacent to duplexes were ligated using T4 RNA ligase 1.

    Ligation:

    Article Title: High-throughput determination of RNA structure by proximity ligation
    Article Snippet: .. Following end-repair, complexes were immediately transferred to 450 uL ligation reaction mix (50 uL 10X T4 DNA Ligase Buffer w/ 10 mM ATP (NEB); 5 uL SuperASE-In (Ambion), 12.5 uL T4 RNA Ligase I (NEB), 382.5 uL 1X PBS w/ 0.2% IGEPAL), and incubated overnight in a 16 °C water bath, after which complexes were added to 1.5 mL TriZOL (Ambion). .. Samples were then purified using Direct-ZOL spin columns (Zymo) according to manufacturer's protocols.

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    New England Biolabs t4 rna ligase 1
    ( a ) Schematic illustration of the high efficiency, purification- and template-free, adapter oligonucleotide adenylation method using <t>T4</t> RNA ligase 1. The 3′ end of the adapter oligo was blocked by –ddC modification to prevent circularization and concatemerization. The 5′ base (shown in black) was swapped between dA, dC, dG, dT, rA, rC, rG, and rU to test bias. ( b ) The adapter adenylation efficiency was investigated as a function of 5′ terminal nucleotide. The reaction conditions were modified to exaggerate differences in efficiency (10 μL volume, 100 units ligase per nanomole adapter, 0.1 nanomole adapter, 30% PEG, 1 hour incubation). The rC and dG adapters are the most and least efficiently adenylated, respectively. ( c ) The adapter adenylation efficiency was then measured as a function of PEG % for a few representative adapters. In all cases, efficiency monotonically increased with PEG %. ( d ) Comparison of adenylation efficiency of as a function of PEG % under standard reaction conditions using the rA and dA adapters. Both the dA and rA adapters are efficiently adenylated at 35% PEG.
    T4 Rna Ligase 1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 203 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ( a ) Schematic illustration of the high efficiency, purification- and template-free, adapter oligonucleotide adenylation method using T4 RNA ligase 1. The 3′ end of the adapter oligo was blocked by –ddC modification to prevent circularization and concatemerization. The 5′ base (shown in black) was swapped between dA, dC, dG, dT, rA, rC, rG, and rU to test bias. ( b ) The adapter adenylation efficiency was investigated as a function of 5′ terminal nucleotide. The reaction conditions were modified to exaggerate differences in efficiency (10 μL volume, 100 units ligase per nanomole adapter, 0.1 nanomole adapter, 30% PEG, 1 hour incubation). The rC and dG adapters are the most and least efficiently adenylated, respectively. ( c ) The adapter adenylation efficiency was then measured as a function of PEG % for a few representative adapters. In all cases, efficiency monotonically increased with PEG %. ( d ) Comparison of adenylation efficiency of as a function of PEG % under standard reaction conditions using the rA and dA adapters. Both the dA and rA adapters are efficiently adenylated at 35% PEG.

    Journal: Scientific Reports

    Article Title: Efficient synthesis of stably adenylated DNA and RNA adapters for microRNA capture using T4 RNA ligase 1

    doi: 10.1038/srep15620

    Figure Lengend Snippet: ( a ) Schematic illustration of the high efficiency, purification- and template-free, adapter oligonucleotide adenylation method using T4 RNA ligase 1. The 3′ end of the adapter oligo was blocked by –ddC modification to prevent circularization and concatemerization. The 5′ base (shown in black) was swapped between dA, dC, dG, dT, rA, rC, rG, and rU to test bias. ( b ) The adapter adenylation efficiency was investigated as a function of 5′ terminal nucleotide. The reaction conditions were modified to exaggerate differences in efficiency (10 μL volume, 100 units ligase per nanomole adapter, 0.1 nanomole adapter, 30% PEG, 1 hour incubation). The rC and dG adapters are the most and least efficiently adenylated, respectively. ( c ) The adapter adenylation efficiency was then measured as a function of PEG % for a few representative adapters. In all cases, efficiency monotonically increased with PEG %. ( d ) Comparison of adenylation efficiency of as a function of PEG % under standard reaction conditions using the rA and dA adapters. Both the dA and rA adapters are efficiently adenylated at 35% PEG.

    Article Snippet: Unless otherwise indicated, the adenylation reaction was performed using the optimized conditions of a 25 μL reaction volume containing 0.05 nanomole dA adapter, 1X T4 RNA Ligase Buffer (New England Biolabs, Ipswich, MA), 35% PEG, 1 mM ATP, and 300 units of T4 RNA Ligase 1 (New England Biolabs, Ipswich, MA) per nanomole adapter.

    Techniques: Purification, Modification, Incubation

    microRNA-adapter ligation was performed using adenylated adapters generated by either (a) T4 RNA ligase 1 or (c) archaeal RNA ligase. The adapters were labeled with Cy5 while the synthetic microRNA were labeled with Cy3. Lanes 1 and 2 show that both methods are capable of fully adenylating the adapters. Lanes 4 and 6 show that let-7a microRNA can be effectively ligated both in the absence and presence of total RNA background. Lane 5 shows that large RNA molecules within the total RNA are captured by both adapters. No de-adenylation is observed with either method. ( b ) The T4 RNA ligase 1 adenylated adapter was used to capture RNA from 10, 100, or 1000 ng of pancreatic tissue total RNA spiked with 0.01 picomoles of 6 synthetic microRNA. The three ligation products from the top are large RNA molecules intrinsic to the total RNA that have been captured by the adapter. As expected, they vary in linear proportion to the total RNA input. The band in the middle is the spiked microRNA captured by the adapter which remains constant across all three samples as expected. The large band at the bottom of the gel is free adenylated Cy5 adapter.

    Journal: Scientific Reports

    Article Title: Efficient synthesis of stably adenylated DNA and RNA adapters for microRNA capture using T4 RNA ligase 1

    doi: 10.1038/srep15620

    Figure Lengend Snippet: microRNA-adapter ligation was performed using adenylated adapters generated by either (a) T4 RNA ligase 1 or (c) archaeal RNA ligase. The adapters were labeled with Cy5 while the synthetic microRNA were labeled with Cy3. Lanes 1 and 2 show that both methods are capable of fully adenylating the adapters. Lanes 4 and 6 show that let-7a microRNA can be effectively ligated both in the absence and presence of total RNA background. Lane 5 shows that large RNA molecules within the total RNA are captured by both adapters. No de-adenylation is observed with either method. ( b ) The T4 RNA ligase 1 adenylated adapter was used to capture RNA from 10, 100, or 1000 ng of pancreatic tissue total RNA spiked with 0.01 picomoles of 6 synthetic microRNA. The three ligation products from the top are large RNA molecules intrinsic to the total RNA that have been captured by the adapter. As expected, they vary in linear proportion to the total RNA input. The band in the middle is the spiked microRNA captured by the adapter which remains constant across all three samples as expected. The large band at the bottom of the gel is free adenylated Cy5 adapter.

    Article Snippet: Unless otherwise indicated, the adenylation reaction was performed using the optimized conditions of a 25 μL reaction volume containing 0.05 nanomole dA adapter, 1X T4 RNA Ligase Buffer (New England Biolabs, Ipswich, MA), 35% PEG, 1 mM ATP, and 300 units of T4 RNA Ligase 1 (New England Biolabs, Ipswich, MA) per nanomole adapter.

    Techniques: Ligation, Generated, Labeling

    Adenylated adapters generated using either T4 RNA ligase 1 or archaeal RNA ligase were used for microRNA-adapter ligation of a mixture containing 80 nt let-7a precursor DNA molecules and 22 nt let-7a mature microRNA molecules. The amount of PEG in the reaction mixture was also varied. Circularized DNA ligation product is only generated using the archaeal RNA ligase adenylated adapters.

    Journal: Scientific Reports

    Article Title: Efficient synthesis of stably adenylated DNA and RNA adapters for microRNA capture using T4 RNA ligase 1

    doi: 10.1038/srep15620

    Figure Lengend Snippet: Adenylated adapters generated using either T4 RNA ligase 1 or archaeal RNA ligase were used for microRNA-adapter ligation of a mixture containing 80 nt let-7a precursor DNA molecules and 22 nt let-7a mature microRNA molecules. The amount of PEG in the reaction mixture was also varied. Circularized DNA ligation product is only generated using the archaeal RNA ligase adenylated adapters.

    Article Snippet: Unless otherwise indicated, the adenylation reaction was performed using the optimized conditions of a 25 μL reaction volume containing 0.05 nanomole dA adapter, 1X T4 RNA Ligase Buffer (New England Biolabs, Ipswich, MA), 35% PEG, 1 mM ATP, and 300 units of T4 RNA Ligase 1 (New England Biolabs, Ipswich, MA) per nanomole adapter.

    Techniques: Generated, Ligation, DNA Ligation

    Schematic overview of the modified protocol. a , wet experiment. Irradiated with 365 nm UV, RNAs were cross-linked by AMT at the paired region, and survive DNase I, RNase T1 and RNase H treatments which digest DNA and single strand RNA. Cross-linked RNAs were ligated by T4 RNA ligase 1. After photoreversal of cross-linkages by 254 nm UV, the ligated RNAs could be sequenced and identified. b , bioinformatics analysis

    Journal: BMC Genomics

    Article Title: Detecting RNA-RNA interactions in E. coli using a modified CLASH method

    doi: 10.1186/s12864-017-3725-3

    Figure Lengend Snippet: Schematic overview of the modified protocol. a , wet experiment. Irradiated with 365 nm UV, RNAs were cross-linked by AMT at the paired region, and survive DNase I, RNase T1 and RNase H treatments which digest DNA and single strand RNA. Cross-linked RNAs were ligated by T4 RNA ligase 1. After photoreversal of cross-linkages by 254 nm UV, the ligated RNAs could be sequenced and identified. b , bioinformatics analysis

    Article Snippet: Cross-linked RNA molecules were then ligated using 40 U of T4 RNA ligase 1 (New England Biolabs, M0204), 1 mM ATP, and 40 U RNase inhibitors in RNA ligase 1 buffer for 1 h at 15 °C, and kept for 16 h at 4 °C.

    Techniques: Modification, Irradiation

    Deadenylation activity of T4 RNA ligase 2 truncated mutants . 5'-adenylated DNA adapters were incubated with an excess of ligase (13.8 pmol), and 12.5% PEG 8000 at 16°C overnight. Oligonucleotide substrates are depicted schematically above the gel. The contents of each sample were resolved on denaturing 15% acrylamide gels and stained with SYBR Gold to visualize nucleic acid. Deadenylation of the DNA adapter (loss of 5'-App) is indicated by a band shift of ~1 nt towards the bottom of the gel. Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2 +MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP.

    Journal: BMC Biotechnology

    Article Title: T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis

    doi: 10.1186/1472-6750-11-72

    Figure Lengend Snippet: Deadenylation activity of T4 RNA ligase 2 truncated mutants . 5'-adenylated DNA adapters were incubated with an excess of ligase (13.8 pmol), and 12.5% PEG 8000 at 16°C overnight. Oligonucleotide substrates are depicted schematically above the gel. The contents of each sample were resolved on denaturing 15% acrylamide gels and stained with SYBR Gold to visualize nucleic acid. Deadenylation of the DNA adapter (loss of 5'-App) is indicated by a band shift of ~1 nt towards the bottom of the gel. Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2 +MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP.

    Article Snippet: T4 RNA ligase 1, T4 RNA ligase 2, T4 RNA ligase 2 Truncated and, T4 RNA ligase 2 Truncated K227Q were obtained from New England Biolabs.

    Techniques: Activity Assay, Incubation, Staining, Electrophoretic Mobility Shift Assay, Binding Assay

    Assaying the formation of side products by T4 RNA ligases . Intermolecular strand-joining reactions containing 5'-adenylated adapters, 21-mer 5'-PO 4 RNA acceptors, and ligase (1 pmol) were incubated at 16°C overnight in the presence of 12.5% PEG 8000. Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA. Products of the reaction were resolved on denaturing 15% acrylamide gels and stained with SYBR Gold. The bands corresponding to the input nucleic acids, the DNA adapter/RNA acceptor ligation product (39 bases), and larger side products are indicated. Ladder = size standard ladder, Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2 +MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP.

    Journal: BMC Biotechnology

    Article Title: T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis

    doi: 10.1186/1472-6750-11-72

    Figure Lengend Snippet: Assaying the formation of side products by T4 RNA ligases . Intermolecular strand-joining reactions containing 5'-adenylated adapters, 21-mer 5'-PO 4 RNA acceptors, and ligase (1 pmol) were incubated at 16°C overnight in the presence of 12.5% PEG 8000. Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA. Products of the reaction were resolved on denaturing 15% acrylamide gels and stained with SYBR Gold. The bands corresponding to the input nucleic acids, the DNA adapter/RNA acceptor ligation product (39 bases), and larger side products are indicated. Ladder = size standard ladder, Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2 +MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP.

    Article Snippet: T4 RNA ligase 1, T4 RNA ligase 2, T4 RNA ligase 2 Truncated and, T4 RNA ligase 2 Truncated K227Q were obtained from New England Biolabs.

    Techniques: Incubation, Staining, Ligation, Binding Assay

    Following AMP during ligation reactions with T4 RNA ligases . (A) 22-mer DNA adapters were 5'-adenylated with α- 32 P-labeled ATP (see materials and methods). Intermolecular strand-joining reactions containing 10 pmol radiolabeled DNA adapter, 5 pmol 21-mer 5'-PO 4 RNA acceptor, and ligase (1 pmol) were incubated overnight at 16°C in the presence of PEG 8000. Reaction products were resolved on a denaturing 15% acrylamide gel and radioactive molecules were visualized by exposure to Phosphor screens. The resulting products were either free AMP in solution (AMP*) or the adapter remaining adenylated (Ap*p-DNA). Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA. P* denotes 32 P-phosphate. (B) Determining the fate of AMP upon T4 RNA ligase-dependent deadenylation. Reactions containing radiolabeled DNA adapter (10 pmol) and ligase (14 pmol) were incubated overnight at 16°C in the presence of 12.5% PEG 8000. Oligonucleotide substrates are depicted schematically above the gel. P* denotes 32 P-phosphate. Reaction products were resolved and visualized as in (A). The resulting products were either free AMP in solution (AMP*), the adapter remaining adenylated (Ap*p-DNA), or AMP covalently bound to the ligase (AMP*-ligase). The lane labeled input contains only Ap*p-DNA. (C) Reactions identical to those in (B) were treated with Proteinase K prior to gel electrophoresis and detection. (D) Reactions containing 10 pmol radiolabeled DNA adapter, 5 pmol 28-mer [5'-PO 4 , 3'-blocked] RNA acceptor, and ligase (1 pmol) were incubated, resolved and detected as in (A). The resulting products were either free AMP in solution (AMP*), adenylated adapter (Ap*p-DNA), or Ap*p-28-mer RNA. The lane labeled RNA size control contains 5'- 32 PO 4 RNA, and the lane labeled input contains only Ap*p-DNA. Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA. P* denotes 32 P-phosphate. In all panels, Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2 +MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP.

    Journal: BMC Biotechnology

    Article Title: T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis

    doi: 10.1186/1472-6750-11-72

    Figure Lengend Snippet: Following AMP during ligation reactions with T4 RNA ligases . (A) 22-mer DNA adapters were 5'-adenylated with α- 32 P-labeled ATP (see materials and methods). Intermolecular strand-joining reactions containing 10 pmol radiolabeled DNA adapter, 5 pmol 21-mer 5'-PO 4 RNA acceptor, and ligase (1 pmol) were incubated overnight at 16°C in the presence of PEG 8000. Reaction products were resolved on a denaturing 15% acrylamide gel and radioactive molecules were visualized by exposure to Phosphor screens. The resulting products were either free AMP in solution (AMP*) or the adapter remaining adenylated (Ap*p-DNA). Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA. P* denotes 32 P-phosphate. (B) Determining the fate of AMP upon T4 RNA ligase-dependent deadenylation. Reactions containing radiolabeled DNA adapter (10 pmol) and ligase (14 pmol) were incubated overnight at 16°C in the presence of 12.5% PEG 8000. Oligonucleotide substrates are depicted schematically above the gel. P* denotes 32 P-phosphate. Reaction products were resolved and visualized as in (A). The resulting products were either free AMP in solution (AMP*), the adapter remaining adenylated (Ap*p-DNA), or AMP covalently bound to the ligase (AMP*-ligase). The lane labeled input contains only Ap*p-DNA. (C) Reactions identical to those in (B) were treated with Proteinase K prior to gel electrophoresis and detection. (D) Reactions containing 10 pmol radiolabeled DNA adapter, 5 pmol 28-mer [5'-PO 4 , 3'-blocked] RNA acceptor, and ligase (1 pmol) were incubated, resolved and detected as in (A). The resulting products were either free AMP in solution (AMP*), adenylated adapter (Ap*p-DNA), or Ap*p-28-mer RNA. The lane labeled RNA size control contains 5'- 32 PO 4 RNA, and the lane labeled input contains only Ap*p-DNA. Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA. P* denotes 32 P-phosphate. In all panels, Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2 +MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP.

    Article Snippet: T4 RNA ligase 1, T4 RNA ligase 2, T4 RNA ligase 2 Truncated and, T4 RNA ligase 2 Truncated K227Q were obtained from New England Biolabs.

    Techniques: Ligation, Labeling, Incubation, Acrylamide Gel Assay, Nucleic Acid Electrophoresis, Binding Assay

    Production of ligation side products by T4 RNA ligases . Intermolecular ligation reactions containing 5'-adenylated DNA adapters, 21-mer 5'-PO 4 RNA acceptors and ligase (1 pmol) were incubated at 16°C overnight with 12.5% PEG 8000. Products of the reactions were resolved on denaturing 15% acrylamide gels and stained with SYBR Gold. The bands corresponding to the input nucleic acids, the DNA adapter/RNA acceptor ligation product (39 bases), and larger side products are indicated. Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA.

    Journal: BMC Biotechnology

    Article Title: T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis

    doi: 10.1186/1472-6750-11-72

    Figure Lengend Snippet: Production of ligation side products by T4 RNA ligases . Intermolecular ligation reactions containing 5'-adenylated DNA adapters, 21-mer 5'-PO 4 RNA acceptors and ligase (1 pmol) were incubated at 16°C overnight with 12.5% PEG 8000. Products of the reactions were resolved on denaturing 15% acrylamide gels and stained with SYBR Gold. The bands corresponding to the input nucleic acids, the DNA adapter/RNA acceptor ligation product (39 bases), and larger side products are indicated. Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA.

    Article Snippet: T4 RNA ligase 1, T4 RNA ligase 2, T4 RNA ligase 2 Truncated and, T4 RNA ligase 2 Truncated K227Q were obtained from New England Biolabs.

    Techniques: Ligation, Incubation, Staining, Binding Assay

    Effect of pH on ligase intermolecular strand-joining activity . (A-D) Intermolecular strand-joining reactions were carried out with 10 pmol 5'-adenylated 17mer DNA, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, and ligase (1 pmol) for 1 hour at 25°C to assess the effect of pH on ligation efficiency. Ligation efficiency was determined by resolving the material in the reactions on denaturing 15% acrylamide gels and quantifying the amount of ligation product versus input nucleic acid. (E-H) Intermolecular strand-joining reactions were carried out with 10 pmol 5'-adenylated 17-mer DNA, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, and ligase (13.8 pmol) for 1 hour at 25°C to assess the effect of pH on ligation efficiency. Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP. Data are shown as the mean +/- SEM of at least three independent experiments.

    Journal: BMC Biotechnology

    Article Title: T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis

    doi: 10.1186/1472-6750-11-72

    Figure Lengend Snippet: Effect of pH on ligase intermolecular strand-joining activity . (A-D) Intermolecular strand-joining reactions were carried out with 10 pmol 5'-adenylated 17mer DNA, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, and ligase (1 pmol) for 1 hour at 25°C to assess the effect of pH on ligation efficiency. Ligation efficiency was determined by resolving the material in the reactions on denaturing 15% acrylamide gels and quantifying the amount of ligation product versus input nucleic acid. (E-H) Intermolecular strand-joining reactions were carried out with 10 pmol 5'-adenylated 17-mer DNA, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, and ligase (13.8 pmol) for 1 hour at 25°C to assess the effect of pH on ligation efficiency. Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP. Data are shown as the mean +/- SEM of at least three independent experiments.

    Article Snippet: T4 RNA ligase 1, T4 RNA ligase 2, T4 RNA ligase 2 Truncated and, T4 RNA ligase 2 Truncated K227Q were obtained from New England Biolabs.

    Techniques: Activity Assay, Labeling, Ligation, Binding Assay

    Analysis of intermolecular strand-joining over time . Strand-joining reactions were carried out with 10 pmol 5'-adenylated adapter, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, and ligase (1 pmol) over a span of 24 hours at 25°C to assess the progress of ligation reactions. Ligation efficiency was determined by resolving the material in the reactions on denaturing 15% acrylamide gels and quantifying the amount of ligation product versus input nucleic acid. Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP. Data are shown as the mean +/- SEM of at least three independent experiments.

    Journal: BMC Biotechnology

    Article Title: T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis

    doi: 10.1186/1472-6750-11-72

    Figure Lengend Snippet: Analysis of intermolecular strand-joining over time . Strand-joining reactions were carried out with 10 pmol 5'-adenylated adapter, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, and ligase (1 pmol) over a span of 24 hours at 25°C to assess the progress of ligation reactions. Ligation efficiency was determined by resolving the material in the reactions on denaturing 15% acrylamide gels and quantifying the amount of ligation product versus input nucleic acid. Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP. Data are shown as the mean +/- SEM of at least three independent experiments.

    Article Snippet: T4 RNA ligase 1, T4 RNA ligase 2, T4 RNA ligase 2 Truncated and, T4 RNA ligase 2 Truncated K227Q were obtained from New England Biolabs.

    Techniques: Labeling, Ligation, Binding Assay

    Identification of leaderless genomic RNA during BCoV persistent infection. (A) Strategy to identify positive-strand leaderless genomic RNA. Poly(A)-containing RNA was selected from total cellular RNA extracted from BCoV-persistently infected cells, treated with alkaline phosphatase, decapped with tobacco acid pyrophosphatase, head-to-tail ligated with T4 RNA ligase I, and used as the template for RT-PCR with the BCoV 5′ UTR-(+)-strand-specific primer 2: BCV107(+) (for RT) and BCoV 3′ UTR-(−)-strand-specific primer 1: BCV3′UTR1(−). (B) RT-PCR product synthesized by the method described in Fig. 1A. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) and with a size of less than 200 bp (lanes 6–7, marked with white arrowhead) were observed. (C) The upper panel shows part of the first 88-nt sequence of the 5′ UTR in the positive-strand BCoV genomic RNA. The positions (1 and 70) are given on the top of the sequence, and the intergenic sequence (IS) UCUAAAC is underlined. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of more than 200 bp from lane 7, as indicated with a black arrowhead in Fig. 1B. (D) The upper panel shows the sequence of the 5′UTR on the positive-strand BCoV genomic RNA, which lacks the first 69 nts; position 70 is given on the top of the sequence. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of less than 200 bp from lane 7, as indicated with a white arrowhead in Fig. 1B. (E) Control reactions to determine if the positive-strand leaderless genome is a degradation product. RT-PCR product was synthesized by the method described in Fig. 1A except RNA sample was not treated with alkaline phosphatase and tobacco acid pyrophosphatase. RT-PCR products with a length of more than 200 bp were detected. (F) Identification of negative-strand leaderless genomic RNA. Total cellular RNA was treated with tobacco acid pyrophosphatase and ligated with T4 RNA ligase I. RT-PCR product was synthesized by the method described in Fig. 1A except that primer BCV3′UTR1(−) was used for RT. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) were observed. M, ds DNA size markers in nt pairs. dpi: days postinfection.

    Journal: PLoS ONE

    Article Title: A Leaderless Genome Identified during Persistent Bovine Coronavirus Infection Is Associated with Attenuation of Gene Expression

    doi: 10.1371/journal.pone.0082176

    Figure Lengend Snippet: Identification of leaderless genomic RNA during BCoV persistent infection. (A) Strategy to identify positive-strand leaderless genomic RNA. Poly(A)-containing RNA was selected from total cellular RNA extracted from BCoV-persistently infected cells, treated with alkaline phosphatase, decapped with tobacco acid pyrophosphatase, head-to-tail ligated with T4 RNA ligase I, and used as the template for RT-PCR with the BCoV 5′ UTR-(+)-strand-specific primer 2: BCV107(+) (for RT) and BCoV 3′ UTR-(−)-strand-specific primer 1: BCV3′UTR1(−). (B) RT-PCR product synthesized by the method described in Fig. 1A. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) and with a size of less than 200 bp (lanes 6–7, marked with white arrowhead) were observed. (C) The upper panel shows part of the first 88-nt sequence of the 5′ UTR in the positive-strand BCoV genomic RNA. The positions (1 and 70) are given on the top of the sequence, and the intergenic sequence (IS) UCUAAAC is underlined. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of more than 200 bp from lane 7, as indicated with a black arrowhead in Fig. 1B. (D) The upper panel shows the sequence of the 5′UTR on the positive-strand BCoV genomic RNA, which lacks the first 69 nts; position 70 is given on the top of the sequence. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of less than 200 bp from lane 7, as indicated with a white arrowhead in Fig. 1B. (E) Control reactions to determine if the positive-strand leaderless genome is a degradation product. RT-PCR product was synthesized by the method described in Fig. 1A except RNA sample was not treated with alkaline phosphatase and tobacco acid pyrophosphatase. RT-PCR products with a length of more than 200 bp were detected. (F) Identification of negative-strand leaderless genomic RNA. Total cellular RNA was treated with tobacco acid pyrophosphatase and ligated with T4 RNA ligase I. RT-PCR product was synthesized by the method described in Fig. 1A except that primer BCV3′UTR1(−) was used for RT. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) were observed. M, ds DNA size markers in nt pairs. dpi: days postinfection.

    Article Snippet: To determine the terminal sequence of viral negative-strand genomic RNA and sgmRNA, total cellular RNA was treated with tobacco acid pyrophosphatase (Epicentre), ligated with T4 RNA ligase I (New England Biolabs) and primer 1: BCV3′UTR1(−) was used for RT; for PCR, primers BCV3′UTR(−) and BCV107(+), and primers BCV3′UTR(−) and RYN(+) were used for determining terminal sequence of negative-strand genomic RNA and subgenomic mRNA, respectively.

    Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Synthesized, Sequencing, Clone Assay