t4 rna ligase 2  (New England Biolabs)


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    Name:
    T4 RNA Ligase 2 truncated
    Description:
    T4 RNA Ligase 2 truncated 10 000 units
    Catalog Number:
    m0242l
    Price:
    283
    Size:
    10 000 units
    Category:
    RNA Ligases
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    New England Biolabs t4 rna ligase 2
    T4 RNA Ligase 2 truncated
    T4 RNA Ligase 2 truncated 10 000 units
    https://www.bioz.com/result/t4 rna ligase 2/product/New England Biolabs
    Average 99 stars, based on 205 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase 2 - by Bioz Stars, 2020-10
    99/100 stars

    Images

    1) Product Images from "Uridylation by TUT4/7 Restricts Retrotransposition of Human LINE-1s"

    Article Title: Uridylation by TUT4/7 Restricts Retrotransposition of Human LINE-1s

    Journal: Cell

    doi: 10.1016/j.cell.2018.07.022

    Graphical Visualization of the 3′ RACE-Seq Approach, Related to Figure 2 (A) Graphical representation of 3′ RACE-seq library preparation and the oligonucleotides used. First, the 3′ adaptor RA3_15N was joined to the 3′ end of RNA by enzymatic ligation. The adaptor has: (i) 5′ rApp modification for efficient and specific ligation by the truncated T4 RNA ligase 2, (ii) delimiter sequence to be used in bioinformatics analyses to exclude RT and PCR artifacts (CTGAC, highlighted in violet), (iii) unique 15N barcode for individual transcript barcoding (highlighted in green), (iv) anchor sequence to pair with the reverse transcription primer (underlined) and (v) dideoxyC on the 3′ end to prevent concatamer formation. The RNA ligated to the adaptor sequence was purified from excess adaptor and reverse transcription was performed with the RT primer, which is compatible with Illumina sequencing and has: (i) sequences to base-pair with the adaptor (underlined), (ii) 6-nucleotide barcode for sample barcoding (highlighted in red), (iii) sequences that base pair with the universal outer primer for nested PCR (blue). Libraries were generated by nested PCR with 2 outer forward primers (F1 and F2) and a single universal reverse primer (uni rev). PCR amplicons of first and second PCRs were purified from excess primers on AmPure beads (Agencourt) before beginning the next step. (B) Flowchart of the bioinformatics approach to 3′ RACE-seq data analysis. The procedure starts at the top. Datasets are shown in rectangles. Software used is depicted in hexagons.
    Figure Legend Snippet: Graphical Visualization of the 3′ RACE-Seq Approach, Related to Figure 2 (A) Graphical representation of 3′ RACE-seq library preparation and the oligonucleotides used. First, the 3′ adaptor RA3_15N was joined to the 3′ end of RNA by enzymatic ligation. The adaptor has: (i) 5′ rApp modification for efficient and specific ligation by the truncated T4 RNA ligase 2, (ii) delimiter sequence to be used in bioinformatics analyses to exclude RT and PCR artifacts (CTGAC, highlighted in violet), (iii) unique 15N barcode for individual transcript barcoding (highlighted in green), (iv) anchor sequence to pair with the reverse transcription primer (underlined) and (v) dideoxyC on the 3′ end to prevent concatamer formation. The RNA ligated to the adaptor sequence was purified from excess adaptor and reverse transcription was performed with the RT primer, which is compatible with Illumina sequencing and has: (i) sequences to base-pair with the adaptor (underlined), (ii) 6-nucleotide barcode for sample barcoding (highlighted in red), (iii) sequences that base pair with the universal outer primer for nested PCR (blue). Libraries were generated by nested PCR with 2 outer forward primers (F1 and F2) and a single universal reverse primer (uni rev). PCR amplicons of first and second PCRs were purified from excess primers on AmPure beads (Agencourt) before beginning the next step. (B) Flowchart of the bioinformatics approach to 3′ RACE-seq data analysis. The procedure starts at the top. Datasets are shown in rectangles. Software used is depicted in hexagons.

    Techniques Used: Ligation, Modification, Sequencing, Polymerase Chain Reaction, Purification, Nested PCR, Generated, Software

    2) 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

    3) Product Images from "A fully enzymatic method for site-directed spin labeling of long RNA"

    Article Title: A fully enzymatic method for site-directed spin labeling of long RNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku553

    Synthesis of site-specific spin-labeled RNA. (A) Native gel electrophoresis on 10% polyacrylamide. RNA segments and DNA splint were loaded in the T4 RNA ligase 2 buffer: lane 1, (pG24 6T -C55); lane 2, (pppG1-G23); lane 3, DNA splint; lane 4, (pppG1-G23) + (pG24 6T -C55) + DNA splint. (B) Denaturing 12% polyacrylamide gel. Lane 1: RNA fragment (G1-G23) acceptor; lane 2: RNA fragment (pG24 6T -C55) donor; lane 3: DNA splint (43-nt); lane 4: preparative ligation; lane 5: purified ligation product. (C) Imino-proton region of 1D spectra recorded at 20°C of the wild-type RNA full-length (top) and the ligation product (bottom). All imino protons were assigned via sequential Nuclear Overhauser Effects (NOEs) observed in 2D-NOESY experiments, with the exception of the resonances at 10.37, 11.09 and 11.43 p.p.m. that could not be identified unambiguously.
    Figure Legend Snippet: Synthesis of site-specific spin-labeled RNA. (A) Native gel electrophoresis on 10% polyacrylamide. RNA segments and DNA splint were loaded in the T4 RNA ligase 2 buffer: lane 1, (pG24 6T -C55); lane 2, (pppG1-G23); lane 3, DNA splint; lane 4, (pppG1-G23) + (pG24 6T -C55) + DNA splint. (B) Denaturing 12% polyacrylamide gel. Lane 1: RNA fragment (G1-G23) acceptor; lane 2: RNA fragment (pG24 6T -C55) donor; lane 3: DNA splint (43-nt); lane 4: preparative ligation; lane 5: purified ligation product. (C) Imino-proton region of 1D spectra recorded at 20°C of the wild-type RNA full-length (top) and the ligation product (bottom). All imino protons were assigned via sequential Nuclear Overhauser Effects (NOEs) observed in 2D-NOESY experiments, with the exception of the resonances at 10.37, 11.09 and 11.43 p.p.m. that could not be identified unambiguously.

    Techniques Used: Labeling, Nucleic Acid Electrophoresis, Ligation, Purification

    4) 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

    5) Product Images from "Highly specific imaging of mRNA in single cells by target RNA-initiated rolling circle amplification specific imaging of mRNA in single cells by target RNA-initiated rolling circle amplification †Electronic supplementary information (ESI) available: Additional experimental materials, methods, DNA sequences and supplementary figures and tables. See DOI: 10.1039/c7sc00292kClick here for additional data file."

    Article Title: Highly specific imaging of mRNA in single cells by target RNA-initiated rolling circle amplification specific imaging of mRNA in single cells by target RNA-initiated rolling circle amplification †Electronic supplementary information (ESI) available: Additional experimental materials, methods, DNA sequences and supplementary figures and tables. See DOI: 10.1039/c7sc00292kClick here for additional data file.

    Journal: Chemical Science

    doi: 10.1039/c7sc00292k

    Detecting target RNA in vitro using target RNA-initiated RCA with different ligases. (a) Fluorescence emission spectra for the target RNA-initiated RCA reaction carried out using different ligases (Splint R, T4 RNA ligase 2 and T4 DNA ligase) and padlock probes (Target, targeting mRNA ACTB; Random, random padlock probe). (b) The fluorescence intensity of the target RNA-initiated RCA reaction profiled in (a).
    Figure Legend Snippet: Detecting target RNA in vitro using target RNA-initiated RCA with different ligases. (a) Fluorescence emission spectra for the target RNA-initiated RCA reaction carried out using different ligases (Splint R, T4 RNA ligase 2 and T4 DNA ligase) and padlock probes (Target, targeting mRNA ACTB; Random, random padlock probe). (b) The fluorescence intensity of the target RNA-initiated RCA reaction profiled in (a).

    Techniques Used: In Vitro, Fluorescence

    Demonstration of the specificity for mRNA imaging in single cells. (a) The RCA reactions were carried using T4 DNA ligase, T4 RNA ligase 2 and Splint R. Four padlock probes were designed for target mRNA TK1: one perfectly matching with the target sequence of mRNA TK1 (Mis-0), two probes with one (Mis-1) or two (Mis-2) mismatching bases compared to target mRNA TK1 and one random probe (Ran). Inset: frequency histogram of RCA amplicons per cell detected. The right column is the average number of RCA amplicons detected in MCF-7 cells using the padlock probes Mis-0, Mis-1, Mis-2 and Ran; (b and c) detection of a single nucleotide difference in mRNA ACTB in human MCF-7 cells (b) and mouse 4T1 cells (c). Inset: the quantification of the average number of RCA amplicons detected (100 cells counted). The cell nuclei are shown in blue, the RCA amplicons appear as green or red spots, and the cell outlines are marked by a dotted line. Scale bars: 10 μm.
    Figure Legend Snippet: Demonstration of the specificity for mRNA imaging in single cells. (a) The RCA reactions were carried using T4 DNA ligase, T4 RNA ligase 2 and Splint R. Four padlock probes were designed for target mRNA TK1: one perfectly matching with the target sequence of mRNA TK1 (Mis-0), two probes with one (Mis-1) or two (Mis-2) mismatching bases compared to target mRNA TK1 and one random probe (Ran). Inset: frequency histogram of RCA amplicons per cell detected. The right column is the average number of RCA amplicons detected in MCF-7 cells using the padlock probes Mis-0, Mis-1, Mis-2 and Ran; (b and c) detection of a single nucleotide difference in mRNA ACTB in human MCF-7 cells (b) and mouse 4T1 cells (c). Inset: the quantification of the average number of RCA amplicons detected (100 cells counted). The cell nuclei are shown in blue, the RCA amplicons appear as green or red spots, and the cell outlines are marked by a dotted line. Scale bars: 10 μm.

    Techniques Used: Imaging, Sequencing

    Effect of different ligases on the efficiency of mRNA imaging in single cells. (a) Imaging of mRNA ACTB using target RNA-initiated RCA in the MCF-7 cells with different ligases: T4 DNA ligase, T4 RNA ligase 2 and Splint R. In situ RCA was conducted using the padlock probe targeting ACTB and a random padlock probe as control. The cell nuclei are shown in blue, the RCA amplicons appear as green spots, and the cell outlines are marked by a dotted line. Scale bars: 10 μm. Inset: frequency histogram of RCA amplicons per cell detected. (b) Quantification of the average number of RCA amplicons per cell detected in (a).
    Figure Legend Snippet: Effect of different ligases on the efficiency of mRNA imaging in single cells. (a) Imaging of mRNA ACTB using target RNA-initiated RCA in the MCF-7 cells with different ligases: T4 DNA ligase, T4 RNA ligase 2 and Splint R. In situ RCA was conducted using the padlock probe targeting ACTB and a random padlock probe as control. The cell nuclei are shown in blue, the RCA amplicons appear as green spots, and the cell outlines are marked by a dotted line. Scale bars: 10 μm. Inset: frequency histogram of RCA amplicons per cell detected. (b) Quantification of the average number of RCA amplicons per cell detected in (a).

    Techniques Used: Imaging, In Situ

    6) Product Images from "Simple and efficient synthesis of 5? pre-adenylated DNA using thermostable RNA ligase"

    Article Title: Simple and efficient synthesis of 5? pre-adenylated DNA using thermostable RNA ligase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr544

    Ligation of adenylated DNA linker made with MthRnl to RNA. AppDNA17c-NH 2 was ligated to RNA22 (lane 2) and FAM-RNA23 (lane 4) using T4 RNA ligase 2 truncated without ATP as described in ‘Materials and Methods’ section. Lanes 1 and 3 are controls without ligase. Single-stranded RNA size markers (Mr) are included for reference. The products were analyzed by a 15% urea–PAGE.
    Figure Legend Snippet: Ligation of adenylated DNA linker made with MthRnl to RNA. AppDNA17c-NH 2 was ligated to RNA22 (lane 2) and FAM-RNA23 (lane 4) using T4 RNA ligase 2 truncated without ATP as described in ‘Materials and Methods’ section. Lanes 1 and 3 are controls without ligase. Single-stranded RNA size markers (Mr) are included for reference. The products were analyzed by a 15% urea–PAGE.

    Techniques Used: Ligation, Polyacrylamide Gel Electrophoresis

    7) Product Images from "Structure-function analysis of Methanobacterium thermoautotrophicum RNA ligase - engineering a thermostable ATP independent enzyme"

    Article Title: Structure-function analysis of Methanobacterium thermoautotrophicum RNA ligase - engineering a thermostable ATP independent enzyme

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-13-24

    (A) Structural comparison of T4 RNA ligase 2 and archaeal RNA ligase PAB1020 active sites. Two structures were superimposed based on coordinates of pre-bound AMP and ATP homolog (AMPPNP) as well as known conserved amino acids of the ligase active sites. The T4Rnl2 structure is represented in grey and PAB1020 in yellow. The numbers of amino acids in the conserved motifs I-V (in parenthesis) are for T4Rnl2 and PAB1020 ortholog MthRnl, which were determined after sequence alignment of two archaeal enzymes. (B) The sequences of the conserved motifs I and V of MthRnl compared to corresponding motifs in the RNA and DNA ligases as discussed in the text. The listed RNA ligases are from: MthRnl ( Methanobacterium thermoautotrophicum ), PAB1020 ( Pyrococcus abyssi ), TS2126 (bacteriophage Thermus scotoductus ), RM378 (bacteriophage Rhodothermus marinus ), T4Rnl1 and T4Rnl2 (bacteriophage T4), AcNPV ( Autographa californica nucleopolyhedrovirus). And DNA ligases are from: PBCV1 ( Chlorella virus), MthDnl ( Methanobacterium thermoautotrophicum ), VacDnl (Vaccinia virus). The conserved lysines are shown in bold.
    Figure Legend Snippet: (A) Structural comparison of T4 RNA ligase 2 and archaeal RNA ligase PAB1020 active sites. Two structures were superimposed based on coordinates of pre-bound AMP and ATP homolog (AMPPNP) as well as known conserved amino acids of the ligase active sites. The T4Rnl2 structure is represented in grey and PAB1020 in yellow. The numbers of amino acids in the conserved motifs I-V (in parenthesis) are for T4Rnl2 and PAB1020 ortholog MthRnl, which were determined after sequence alignment of two archaeal enzymes. (B) The sequences of the conserved motifs I and V of MthRnl compared to corresponding motifs in the RNA and DNA ligases as discussed in the text. The listed RNA ligases are from: MthRnl ( Methanobacterium thermoautotrophicum ), PAB1020 ( Pyrococcus abyssi ), TS2126 (bacteriophage Thermus scotoductus ), RM378 (bacteriophage Rhodothermus marinus ), T4Rnl1 and T4Rnl2 (bacteriophage T4), AcNPV ( Autographa californica nucleopolyhedrovirus). And DNA ligases are from: PBCV1 ( Chlorella virus), MthDnl ( Methanobacterium thermoautotrophicum ), VacDnl (Vaccinia virus). The conserved lysines are shown in bold.

    Techniques Used: Sequencing

    8) 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

    9) 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

    10) Product Images from "Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture"

    Article Title: Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0094619

    MicroRNA capture was performed with 4 different ligases using the vendor recommended protocols to compare capture efficiency across 20 different microRNA. The ligation products were analyzed by 15% denaturing urea-PAGE. Capture efficiency was determined by performing a Cy3 scan and comparing the intensities of the ∼40 nt captured microRNA band versus the ∼20 nt free microRNA band. T4 RNA Ligase 2 truncated (T4 Rnl2 T) had high average capture efficiency and low bias but many randomly sized background products. The point mutant enzymes T4 RNA Ligase 2 truncated K227Q (T4 Rnl2 TK) and T4 RNA Ligase 2 truncated KQ (T4 Rnl2 TKQ) had decreased side product formation but also lower average capture efficiency and higher bias. Thermostable 5′ App DNA/RNA Ligase (Mth Rnl), which was performed at 65°C instead of 25°C, had similar average capture efficiency and bias but with distinct ligation efficiency pattern.
    Figure Legend Snippet: MicroRNA capture was performed with 4 different ligases using the vendor recommended protocols to compare capture efficiency across 20 different microRNA. The ligation products were analyzed by 15% denaturing urea-PAGE. Capture efficiency was determined by performing a Cy3 scan and comparing the intensities of the ∼40 nt captured microRNA band versus the ∼20 nt free microRNA band. T4 RNA Ligase 2 truncated (T4 Rnl2 T) had high average capture efficiency and low bias but many randomly sized background products. The point mutant enzymes T4 RNA Ligase 2 truncated K227Q (T4 Rnl2 TK) and T4 RNA Ligase 2 truncated KQ (T4 Rnl2 TKQ) had decreased side product formation but also lower average capture efficiency and higher bias. Thermostable 5′ App DNA/RNA Ligase (Mth Rnl), which was performed at 65°C instead of 25°C, had similar average capture efficiency and bias but with distinct ligation efficiency pattern.

    Techniques Used: Ligation, Polyacrylamide Gel Electrophoresis, Mutagenesis

    Schematic illustration of microRNA capture by 3′ adapter ligation. The 19 nt, enzymatically pre-adenlyated adapter is ligated to the 3′ OH of microRNA using T4 RNA ligase 2. The reaction is run at 25°C for 4 hours in the absence of ATP. In order to characterize capture efficiency, the microRNA is end labeled with Cy3. The 3′ end of the adapter is blocked by –ddC, a fluorophore, or other moiety to prevent the formation of concatemers and circularized products.
    Figure Legend Snippet: Schematic illustration of microRNA capture by 3′ adapter ligation. The 19 nt, enzymatically pre-adenlyated adapter is ligated to the 3′ OH of microRNA using T4 RNA ligase 2. The reaction is run at 25°C for 4 hours in the absence of ATP. In order to characterize capture efficiency, the microRNA is end labeled with Cy3. The 3′ end of the adapter is blocked by –ddC, a fluorophore, or other moiety to prevent the formation of concatemers and circularized products.

    Techniques Used: Ligation, Labeling

    11) Product Images from "Simple and efficient synthesis of 5? pre-adenylated DNA using thermostable RNA ligase"

    Article Title: Simple and efficient synthesis of 5? pre-adenylated DNA using thermostable RNA ligase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr544

    Ligation of adenylated DNA linker made with MthRnl to RNA. AppDNA17c-NH 2 was ligated to RNA22 (lane 2) and FAM-RNA23 (lane 4) using T4 RNA ligase 2 truncated without ATP as described in ‘Materials and Methods’ section. Lanes 1 and 3 are controls without ligase. Single-stranded RNA size markers (Mr) are included for reference. The products were analyzed by a 15% urea–PAGE.
    Figure Legend Snippet: Ligation of adenylated DNA linker made with MthRnl to RNA. AppDNA17c-NH 2 was ligated to RNA22 (lane 2) and FAM-RNA23 (lane 4) using T4 RNA ligase 2 truncated without ATP as described in ‘Materials and Methods’ section. Lanes 1 and 3 are controls without ligase. Single-stranded RNA size markers (Mr) are included for reference. The products were analyzed by a 15% urea–PAGE.

    Techniques Used: Ligation, Polyacrylamide Gel Electrophoresis

    12) Product Images from "Rolling Circle Translation of Circular RNA in Living Human Cells"

    Article Title: Rolling Circle Translation of Circular RNA in Living Human Cells

    Journal: Scientific Reports

    doi: 10.1038/srep16435

    Synthesis of circular RNAs. ( A ) A scheme for the synthesis of circular RNAs used in this study. Transcribed linear RNAs were annealed to its complementary DNA oligomer and then ligated using T4 RNA ligase 2 to produce the circular RNA. ( B , C ) Verification of their circularity of the RNAs. The RNAs were incubated with RNase R and the reactions were analysed by denaturing PAGE. The gels were visualised by SYBR Green II staining.
    Figure Legend Snippet: Synthesis of circular RNAs. ( A ) A scheme for the synthesis of circular RNAs used in this study. Transcribed linear RNAs were annealed to its complementary DNA oligomer and then ligated using T4 RNA ligase 2 to produce the circular RNA. ( B , C ) Verification of their circularity of the RNAs. The RNAs were incubated with RNase R and the reactions were analysed by denaturing PAGE. The gels were visualised by SYBR Green II staining.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis, SYBR Green Assay, Staining

    13) Product Images from "Rolling Circle Translation of Circular RNA in Living Human Cells"

    Article Title: Rolling Circle Translation of Circular RNA in Living Human Cells

    Journal: Scientific Reports

    doi: 10.1038/srep16435

    Synthesis of circular RNAs. ( A ) A scheme for the synthesis of circular RNAs used in this study. Transcribed linear RNAs were annealed to its complementary DNA oligomer and then ligated using T4 RNA ligase 2 to produce the circular RNA. ( B , C ) Verification of their circularity of the RNAs. The RNAs were incubated with RNase R and the reactions were analysed by denaturing PAGE. The gels were visualised by SYBR Green II staining.
    Figure Legend Snippet: Synthesis of circular RNAs. ( A ) A scheme for the synthesis of circular RNAs used in this study. Transcribed linear RNAs were annealed to its complementary DNA oligomer and then ligated using T4 RNA ligase 2 to produce the circular RNA. ( B , C ) Verification of their circularity of the RNAs. The RNAs were incubated with RNase R and the reactions were analysed by denaturing PAGE. The gels were visualised by SYBR Green II staining.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis, SYBR Green Assay, Staining

    14) 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

    15) Product Images from "LOTTE-seq (Long hairpin oligonucleotide based tRNA high-throughput sequencing): specific selection of tRNAs with 3’-CCA end for high-throughput sequencing"

    Article Title: LOTTE-seq (Long hairpin oligonucleotide based tRNA high-throughput sequencing): specific selection of tRNAs with 3’-CCA end for high-throughput sequencing

    Journal: RNA Biology

    doi: 10.1080/15476286.2019.1664250

    DNA hairpin adapter ligation. (A) DNA hairpin adapter for LOTTE-seq. The 5ʹ-end of the TGGN overhang is phosphorylated for ligation, the base-paired 3ʹ-end for blocking unwanted side reactions. RT primer binding site is indicated. (B) Adapter ligation catalysed by T4 DNA ligase, T4 RNA ligase 1 and T4 RNA ligase 2 (truncated KQ). Hairpin adapter was incubated with radioactively labelled in vitro transcribed yeast tRNA Phe with and without CCA-end. Only T4 DNA ligase fuses the tRNA with 3ʹ-CCA-end to the adapter hairpin at high selectivity, while RNA ligases 1 and 2 show considerable amounts of side reaction products with the transcript lacking a CCA-end (indicated by asterisks *). T4 RNA ligase 1 shows an additional high molecular weight product migrating in the upper part of the gel, probably resulting from the ligation of two tRNA molecules (**). The panel shows a prolonged exposure of the gels in order to visualize any unspecific ligation side reaction products. In a subsequent PCR-based amplification, such products will represent a considerable unwanted part of the sequence reads. (C) T4 DNA ligase-catalysed hairpin adapter ligation on tRNA transcripts with different 3ʹ-ends. Only the tRNA with a complete 3ʹ-CCA end was accepted for ligation, indicating a high specificity of the adapter ligation for mature tRNA 3ʹ-ends. When the tRNAs with different 3ʹ-ends were pooled, T4 DNA ligase exclusively selects the mature tRNA with CCA-end for ligation.
    Figure Legend Snippet: DNA hairpin adapter ligation. (A) DNA hairpin adapter for LOTTE-seq. The 5ʹ-end of the TGGN overhang is phosphorylated for ligation, the base-paired 3ʹ-end for blocking unwanted side reactions. RT primer binding site is indicated. (B) Adapter ligation catalysed by T4 DNA ligase, T4 RNA ligase 1 and T4 RNA ligase 2 (truncated KQ). Hairpin adapter was incubated with radioactively labelled in vitro transcribed yeast tRNA Phe with and without CCA-end. Only T4 DNA ligase fuses the tRNA with 3ʹ-CCA-end to the adapter hairpin at high selectivity, while RNA ligases 1 and 2 show considerable amounts of side reaction products with the transcript lacking a CCA-end (indicated by asterisks *). T4 RNA ligase 1 shows an additional high molecular weight product migrating in the upper part of the gel, probably resulting from the ligation of two tRNA molecules (**). The panel shows a prolonged exposure of the gels in order to visualize any unspecific ligation side reaction products. In a subsequent PCR-based amplification, such products will represent a considerable unwanted part of the sequence reads. (C) T4 DNA ligase-catalysed hairpin adapter ligation on tRNA transcripts with different 3ʹ-ends. Only the tRNA with a complete 3ʹ-CCA end was accepted for ligation, indicating a high specificity of the adapter ligation for mature tRNA 3ʹ-ends. When the tRNAs with different 3ʹ-ends were pooled, T4 DNA ligase exclusively selects the mature tRNA with CCA-end for ligation.

    Techniques Used: Ligation, Blocking Assay, Binding Assay, Incubation, In Vitro, Molecular Weight, Polymerase Chain Reaction, Amplification, Sequencing

    16) 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

    17) 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

    18) 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

    19) Product Images from "Arm-specific cleavage and mutation during reverse transcription of 2΄,5΄-branched RNA by Moloney murine leukemia virus reverse transcriptase"

    Article Title: Arm-specific cleavage and mutation during reverse transcription of 2΄,5΄-branched RNA by Moloney murine leukemia virus reverse transcriptase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx073

    Scheme of the splinted-ligation method in bRNA construction. In this method, a 2΄-5΄ linked ribo-guanosine (G)-nucleoside in an RNA strand containing the 5΄-segment and 2΄-arm (precursor 1) is transformed into a branchpoint nucleotide by ligation to an RNA strand representing the 3΄-arm (precursor 2). To do so, the two precursors are hybridized partially to a complementary RNA bridge. In this way, the 5΄-phosphate of precursor 2 is brought close to the free 3΄-hydroxyl of the 2΄-5΄ linked nucleoside of precursor 1. The two oligonucleotides are then joined by T4 RNA Ligase 2. Red, blue, and pink symbols ‘w’ represent RNA; the black line represents DNA. The 2΄-5΄ linked ribo-G-nucleoside in precursor 1 at nucleotide (nt) position 37 is highlighted. Nucleic acids downstream of a 2΄-5΄ linkage are plotted vertically in linear and branched oligonucleotides.
    Figure Legend Snippet: Scheme of the splinted-ligation method in bRNA construction. In this method, a 2΄-5΄ linked ribo-guanosine (G)-nucleoside in an RNA strand containing the 5΄-segment and 2΄-arm (precursor 1) is transformed into a branchpoint nucleotide by ligation to an RNA strand representing the 3΄-arm (precursor 2). To do so, the two precursors are hybridized partially to a complementary RNA bridge. In this way, the 5΄-phosphate of precursor 2 is brought close to the free 3΄-hydroxyl of the 2΄-5΄ linked nucleoside of precursor 1. The two oligonucleotides are then joined by T4 RNA Ligase 2. Red, blue, and pink symbols ‘w’ represent RNA; the black line represents DNA. The 2΄-5΄ linked ribo-G-nucleoside in precursor 1 at nucleotide (nt) position 37 is highlighted. Nucleic acids downstream of a 2΄-5΄ linkage are plotted vertically in linear and branched oligonucleotides.

    Techniques Used: Ligation, Transformation Assay

    20) Product Images from "Simple and efficient synthesis of 5? pre-adenylated DNA using thermostable RNA ligase"

    Article Title: Simple and efficient synthesis of 5? pre-adenylated DNA using thermostable RNA ligase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr544

    Ligation of adenylated DNA linker made with MthRnl to RNA. AppDNA17c-NH 2 was ligated to RNA22 (lane 2) and FAM-RNA23 (lane 4) using T4 RNA ligase 2 truncated without ATP as described in ‘Materials and Methods’ section. Lanes 1 and 3 are controls without ligase. Single-stranded RNA size markers (Mr) are included for reference. The products were analyzed by a 15% urea–PAGE.
    Figure Legend Snippet: Ligation of adenylated DNA linker made with MthRnl to RNA. AppDNA17c-NH 2 was ligated to RNA22 (lane 2) and FAM-RNA23 (lane 4) using T4 RNA ligase 2 truncated without ATP as described in ‘Materials and Methods’ section. Lanes 1 and 3 are controls without ligase. Single-stranded RNA size markers (Mr) are included for reference. The products were analyzed by a 15% urea–PAGE.

    Techniques Used: Ligation, Polyacrylamide Gel Electrophoresis

    21) Product Images from "Rolling Circle Translation of Circular RNA in Living Human Cells"

    Article Title: Rolling Circle Translation of Circular RNA in Living Human Cells

    Journal: Scientific Reports

    doi: 10.1038/srep16435

    Synthesis of circular RNAs. ( A ) A scheme for the synthesis of circular RNAs used in this study. Transcribed linear RNAs were annealed to its complementary DNA oligomer and then ligated using T4 RNA ligase 2 to produce the circular RNA. ( B , C ) Verification of their circularity of the RNAs. The RNAs were incubated with RNase R and the reactions were analysed by denaturing PAGE. The gels were visualised by SYBR Green II staining.
    Figure Legend Snippet: Synthesis of circular RNAs. ( A ) A scheme for the synthesis of circular RNAs used in this study. Transcribed linear RNAs were annealed to its complementary DNA oligomer and then ligated using T4 RNA ligase 2 to produce the circular RNA. ( B , C ) Verification of their circularity of the RNAs. The RNAs were incubated with RNase R and the reactions were analysed by denaturing PAGE. The gels were visualised by SYBR Green II staining.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis, SYBR Green Assay, Staining

    22) Product Images from "Monitoring co-transcriptional folding of riboswitches through helicase unwinding"

    Article Title: Monitoring co-transcriptional folding of riboswitches through helicase unwinding

    Journal: Methods in enzymology

    doi: 10.1016/bs.mie.2019.05.031

    Work-flow of duplex preparation. (A) An example helicase unwinding event displaces the T20-DNA strand (black) to allow for the RNA strand (gray and colored) to sequentially fold in a 5’ to 3’ direction. After complete unwinding, only the Cy3-labeled RNA strand and Cy5-labeled biotinylated DNA strand remain. RNA folding should be observed as a decrease in the distance between the Cy3 and Cy5 dyes, increasing FRET efficiency. For riboswitches, folded states may contain mutually exclusive paired regions (pink and green) observed by changes in FRET. (B) The RNA 3’ fragment containing a 3’ region complementary to the biotinylated DNA is in vitro transcribed and purified. After phosphorylation at the 5’- end and purification, the 3’ fragment is ligated with T4 RNA Ligase 2 to the chemically synthesized Cy3-labeled 5’ fragment via annealing to a DNA splint (black). After purification to remove the splint and unligated fragments, the ligated RNA is annealed to a T20-DNA (black) containing the dT20 helicase binding site at the 3’ end and a biotinylated, Cy5-labeled DNA tether for attachment to the streptavidin-coated slide surface.
    Figure Legend Snippet: Work-flow of duplex preparation. (A) An example helicase unwinding event displaces the T20-DNA strand (black) to allow for the RNA strand (gray and colored) to sequentially fold in a 5’ to 3’ direction. After complete unwinding, only the Cy3-labeled RNA strand and Cy5-labeled biotinylated DNA strand remain. RNA folding should be observed as a decrease in the distance between the Cy3 and Cy5 dyes, increasing FRET efficiency. For riboswitches, folded states may contain mutually exclusive paired regions (pink and green) observed by changes in FRET. (B) The RNA 3’ fragment containing a 3’ region complementary to the biotinylated DNA is in vitro transcribed and purified. After phosphorylation at the 5’- end and purification, the 3’ fragment is ligated with T4 RNA Ligase 2 to the chemically synthesized Cy3-labeled 5’ fragment via annealing to a DNA splint (black). After purification to remove the splint and unligated fragments, the ligated RNA is annealed to a T20-DNA (black) containing the dT20 helicase binding site at the 3’ end and a biotinylated, Cy5-labeled DNA tether for attachment to the streptavidin-coated slide surface.

    Techniques Used: Labeling, In Vitro, Purification, Synthesized, Binding Assay

    23) 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

    24) Product Images from "Random-sequence genetic oligomer pools display an innate potential for ligation and recombination"

    Article Title: Random-sequence genetic oligomer pools display an innate potential for ligation and recombination

    Journal: eLife

    doi: 10.7554/eLife.43022

    Analysis of regioselectivity of ligation by H4 and J4 RNA motifs. ( A ) Partial RNAse T1 digest of T4-RNA Ligase 2 (Rnl2) ligated H4 (H4T) (Tab S2) and self-ligated H4T RNA. Regiospecific nuclease-catalysed cleavage at the ligation junction confirms that 3’−5’ linkages were formed in both ligation reactions. Note that cleavage of self-ligated H4T by RNAse T1 is slower due to the presence of the co-purified splint (H4_splintA) RNA used for in-ice H4T ligation. ( B ) A 3’−5’ regioselective 8–17 DNAzyme cleaves a typical self-ligated (H4, lig) or enzymatically ligated (H4, Rnl2) full-length H4 clone from the original semi-random RNA pool at the ligation junction with similar efficiencies. ( C ) A minimal J4 cis motif (J4-min) is unable to cleave an enzymatically produced 3’−5’ ligation site (J4-min Rnl2; lane 1, 2; 15 hr, RT, see Material and methods). In contrast, the same Rnl2 product is efficiently cleaved by a custom DNAzyme (lane 3, E1111_J4) under the same conditions. However, J4-min catalyses reverse cleavage of its own gel-purified in-ice ligation product, suggesting that the ligation reaction of J4 yields RNA with a 2’−5’ phosphodiester linkage. Rapid initial cleavage of self-ligated J4-min that occurs during the mixing dead time is inhibited after annealing of the J4-min RNA to the E1111_J4 DNAzyme, suggesting that the J4 internal loop is a prerequisite for rapid self-cleavage.
    Figure Legend Snippet: Analysis of regioselectivity of ligation by H4 and J4 RNA motifs. ( A ) Partial RNAse T1 digest of T4-RNA Ligase 2 (Rnl2) ligated H4 (H4T) (Tab S2) and self-ligated H4T RNA. Regiospecific nuclease-catalysed cleavage at the ligation junction confirms that 3’−5’ linkages were formed in both ligation reactions. Note that cleavage of self-ligated H4T by RNAse T1 is slower due to the presence of the co-purified splint (H4_splintA) RNA used for in-ice H4T ligation. ( B ) A 3’−5’ regioselective 8–17 DNAzyme cleaves a typical self-ligated (H4, lig) or enzymatically ligated (H4, Rnl2) full-length H4 clone from the original semi-random RNA pool at the ligation junction with similar efficiencies. ( C ) A minimal J4 cis motif (J4-min) is unable to cleave an enzymatically produced 3’−5’ ligation site (J4-min Rnl2; lane 1, 2; 15 hr, RT, see Material and methods). In contrast, the same Rnl2 product is efficiently cleaved by a custom DNAzyme (lane 3, E1111_J4) under the same conditions. However, J4-min catalyses reverse cleavage of its own gel-purified in-ice ligation product, suggesting that the ligation reaction of J4 yields RNA with a 2’−5’ phosphodiester linkage. Rapid initial cleavage of self-ligated J4-min that occurs during the mixing dead time is inhibited after annealing of the J4-min RNA to the E1111_J4 DNAzyme, suggesting that the J4 internal loop is a prerequisite for rapid self-cleavage.

    Techniques Used: Ligation, Purification, Produced

    25) 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

    Effect of PEG 8000 on ligase intermolecular strand-joining activity . Strand-joining reactions were carried out with 10 pmol 5'-adenylated 17-mer DNA, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, ligase (13.8 pmol), and varying amounts of PEG 8000 for 1 hour at 25°C to assess the effect of PEG 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. 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 PEG 8000 on ligase intermolecular strand-joining activity . Strand-joining reactions were carried out with 10 pmol 5'-adenylated 17-mer DNA, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, ligase (13.8 pmol), and varying amounts of PEG 8000 for 1 hour at 25°C to assess the effect of PEG 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. 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

    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

    Purification and activity of T4 RNA Ligase 2 truncated mutants . (A) Aliquots of T4 RNA ligase 2 truncated and mutants were separated on 10-20% Tris-glycine SDS polyacrylamide gels and stained with Coomassie blue. The size (in kDa) of marker polypeptides are indicated on the left. (B) Intermolecular strand-joining activity of T4 RNA ligase 2 truncated mutants under multiple turnover conditions. 10 pmol 5'-adenylated 17-mer DNA was incubated for one hour at 25°C with 5 pmol 5'- FAM-labeled 31-mer RNA. 1 pmol of each ligase was added into reaction mixture. The reaction products were resolved on denaturing 15% acrylamide gels, scanned and quantified as described in the methods section. 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. * denotes difference in means p
    Figure Legend Snippet: Purification and activity of T4 RNA Ligase 2 truncated mutants . (A) Aliquots of T4 RNA ligase 2 truncated and mutants were separated on 10-20% Tris-glycine SDS polyacrylamide gels and stained with Coomassie blue. The size (in kDa) of marker polypeptides are indicated on the left. (B) Intermolecular strand-joining activity of T4 RNA ligase 2 truncated mutants under multiple turnover conditions. 10 pmol 5'-adenylated 17-mer DNA was incubated for one hour at 25°C with 5 pmol 5'- FAM-labeled 31-mer RNA. 1 pmol of each ligase was added into reaction mixture. The reaction products were resolved on denaturing 15% acrylamide gels, scanned and quantified as described in the methods section. 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. * denotes difference in means p

    Techniques Used: Purification, Activity Assay, Staining, Marker, Incubation, Labeling, 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

    26) 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

    Effect of PEG 8000 on ligase intermolecular strand-joining activity . Strand-joining reactions were carried out with 10 pmol 5'-adenylated 17-mer DNA, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, ligase (13.8 pmol), and varying amounts of PEG 8000 for 1 hour at 25°C to assess the effect of PEG 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. 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 PEG 8000 on ligase intermolecular strand-joining activity . Strand-joining reactions were carried out with 10 pmol 5'-adenylated 17-mer DNA, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, ligase (13.8 pmol), and varying amounts of PEG 8000 for 1 hour at 25°C to assess the effect of PEG 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. 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

    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

    Purification and activity of T4 RNA Ligase 2 truncated mutants . (A) Aliquots of T4 RNA ligase 2 truncated and mutants were separated on 10-20% Tris-glycine SDS polyacrylamide gels and stained with Coomassie blue. The size (in kDa) of marker polypeptides are indicated on the left. (B) Intermolecular strand-joining activity of T4 RNA ligase 2 truncated mutants under multiple turnover conditions. 10 pmol 5'-adenylated 17-mer DNA was incubated for one hour at 25°C with 5 pmol 5'- FAM-labeled 31-mer RNA. 1 pmol of each ligase was added into reaction mixture. The reaction products were resolved on denaturing 15% acrylamide gels, scanned and quantified as described in the methods section. 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. * denotes difference in means p
    Figure Legend Snippet: Purification and activity of T4 RNA Ligase 2 truncated mutants . (A) Aliquots of T4 RNA ligase 2 truncated and mutants were separated on 10-20% Tris-glycine SDS polyacrylamide gels and stained with Coomassie blue. The size (in kDa) of marker polypeptides are indicated on the left. (B) Intermolecular strand-joining activity of T4 RNA ligase 2 truncated mutants under multiple turnover conditions. 10 pmol 5'-adenylated 17-mer DNA was incubated for one hour at 25°C with 5 pmol 5'- FAM-labeled 31-mer RNA. 1 pmol of each ligase was added into reaction mixture. The reaction products were resolved on denaturing 15% acrylamide gels, scanned and quantified as described in the methods section. 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. * denotes difference in means p

    Techniques Used: Purification, Activity Assay, Staining, Marker, Incubation, Labeling, 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

    27) Product Images from "Simple and efficient synthesis of 5? pre-adenylated DNA using thermostable RNA ligase"

    Article Title: Simple and efficient synthesis of 5? pre-adenylated DNA using thermostable RNA ligase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr544

    Ligation of adenylated DNA linker made with MthRnl to RNA. AppDNA17c-NH 2 was ligated to RNA22 (lane 2) and FAM-RNA23 (lane 4) using T4 RNA ligase 2 truncated without ATP as described in ‘Materials and Methods’ section. Lanes 1 and 3 are controls without ligase. Single-stranded RNA size markers (Mr) are included for reference. The products were analyzed by a 15% urea–PAGE.
    Figure Legend Snippet: Ligation of adenylated DNA linker made with MthRnl to RNA. AppDNA17c-NH 2 was ligated to RNA22 (lane 2) and FAM-RNA23 (lane 4) using T4 RNA ligase 2 truncated without ATP as described in ‘Materials and Methods’ section. Lanes 1 and 3 are controls without ligase. Single-stranded RNA size markers (Mr) are included for reference. The products were analyzed by a 15% urea–PAGE.

    Techniques Used: Ligation, Polyacrylamide Gel Electrophoresis

    28) Product Images from "Simple and efficient synthesis of 5? pre-adenylated DNA using thermostable RNA ligase"

    Article Title: Simple and efficient synthesis of 5? pre-adenylated DNA using thermostable RNA ligase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr544

    Ligation of adenylated DNA linker made with MthRnl to RNA. AppDNA17c-NH 2 was ligated to RNA22 (lane 2) and FAM-RNA23 (lane 4) using T4 RNA ligase 2 truncated without ATP as described in ‘Materials and Methods’ section. Lanes 1 and 3 are controls without ligase. Single-stranded RNA size markers (Mr) are included for reference. The products were analyzed by a 15% urea–PAGE.
    Figure Legend Snippet: Ligation of adenylated DNA linker made with MthRnl to RNA. AppDNA17c-NH 2 was ligated to RNA22 (lane 2) and FAM-RNA23 (lane 4) using T4 RNA ligase 2 truncated without ATP as described in ‘Materials and Methods’ section. Lanes 1 and 3 are controls without ligase. Single-stranded RNA size markers (Mr) are included for reference. The products were analyzed by a 15% urea–PAGE.

    Techniques Used: Ligation, Polyacrylamide Gel Electrophoresis

    29) 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

    30) Product Images from "Random-sequence genetic oligomer pools display an innate potential for ligation and recombination"

    Article Title: Random-sequence genetic oligomer pools display an innate potential for ligation and recombination

    Journal: eLife

    doi: 10.7554/eLife.43022

    Analysis of regioselectivity of ligation by H4 and J4 RNA motifs. ( A ) Partial RNAse T1 digest of T4-RNA Ligase 2 (Rnl2) ligated H4 (H4T) (Tab S2) and self-ligated H4T RNA. Regiospecific nuclease-catalysed cleavage at the ligation junction confirms that 3’−5’ linkages were formed in both ligation reactions. Note that cleavage of self-ligated H4T by RNAse T1 is slower due to the presence of the co-purified splint (H4_splintA) RNA used for in-ice H4T ligation. ( B ) A 3’−5’ regioselective 8–17 DNAzyme cleaves a typical self-ligated (H4, lig) or enzymatically ligated (H4, Rnl2) full-length H4 clone from the original semi-random RNA pool at the ligation junction with similar efficiencies. ( C ) A minimal J4 cis motif (J4-min) is unable to cleave an enzymatically produced 3’−5’ ligation site (J4-min Rnl2; lane 1, 2; 15 hr, RT, see Material and methods). In contrast, the same Rnl2 product is efficiently cleaved by a custom DNAzyme (lane 3, E1111_J4) under the same conditions. However, J4-min catalyses reverse cleavage of its own gel-purified in-ice ligation product, suggesting that the ligation reaction of J4 yields RNA with a 2’−5’ phosphodiester linkage. Rapid initial cleavage of self-ligated J4-min that occurs during the mixing dead time is inhibited after annealing of the J4-min RNA to the E1111_J4 DNAzyme, suggesting that the J4 internal loop is a prerequisite for rapid self-cleavage.
    Figure Legend Snippet: Analysis of regioselectivity of ligation by H4 and J4 RNA motifs. ( A ) Partial RNAse T1 digest of T4-RNA Ligase 2 (Rnl2) ligated H4 (H4T) (Tab S2) and self-ligated H4T RNA. Regiospecific nuclease-catalysed cleavage at the ligation junction confirms that 3’−5’ linkages were formed in both ligation reactions. Note that cleavage of self-ligated H4T by RNAse T1 is slower due to the presence of the co-purified splint (H4_splintA) RNA used for in-ice H4T ligation. ( B ) A 3’−5’ regioselective 8–17 DNAzyme cleaves a typical self-ligated (H4, lig) or enzymatically ligated (H4, Rnl2) full-length H4 clone from the original semi-random RNA pool at the ligation junction with similar efficiencies. ( C ) A minimal J4 cis motif (J4-min) is unable to cleave an enzymatically produced 3’−5’ ligation site (J4-min Rnl2; lane 1, 2; 15 hr, RT, see Material and methods). In contrast, the same Rnl2 product is efficiently cleaved by a custom DNAzyme (lane 3, E1111_J4) under the same conditions. However, J4-min catalyses reverse cleavage of its own gel-purified in-ice ligation product, suggesting that the ligation reaction of J4 yields RNA with a 2’−5’ phosphodiester linkage. Rapid initial cleavage of self-ligated J4-min that occurs during the mixing dead time is inhibited after annealing of the J4-min RNA to the E1111_J4 DNAzyme, suggesting that the J4 internal loop is a prerequisite for rapid self-cleavage.

    Techniques Used: Ligation, Purification, Produced

    31) Product Images from "Quantitative tRNA-sequencing uncovers metazoan tissue-specific tRNA regulation"

    Article Title: Quantitative tRNA-sequencing uncovers metazoan tissue-specific tRNA regulation

    Journal: Nature Communications

    doi: 10.1038/s41467-020-17879-x

    Quantitative mature tRNA sequencing (QuantM-seq). a Outline of QuantM-seq. tRNA depictions are in black, adapter depictions are in green, and sequences corresponding the RT primer are depicted in blue. The rG and rN at the end of the 5′ AD indicate ribonucleotides. b Polyacrylamide gel showing products and efficiency of adapter ligation onto tRNA. Rnl2: T4 RNA Ligase 2. Asterisk (*) indicates 5S and 5.8S ribosomal RNA bands. c Polyacrylamide gel showing products of reverse transcription (cDNA). Rnl2: T4 RNA Ligase 2. d Images of tRNA arrays; each array represents an independent replicate. For the probes spotted at each position see Source Data . e Scatter plot of reads per million derived from QuantM-seq versus array intensities derived from densitometry with a fitted linear trendline. Shaded area represents the 95% confidence interval of the linear trendline. f Scatter plot of northern blot versus array intensities derived from densitometry with a fitted linear trendline. Shaded area represents the 95% confidence interval of the linear trendline. Source data are provided as a Source Data file for ( b – f ).
    Figure Legend Snippet: Quantitative mature tRNA sequencing (QuantM-seq). a Outline of QuantM-seq. tRNA depictions are in black, adapter depictions are in green, and sequences corresponding the RT primer are depicted in blue. The rG and rN at the end of the 5′ AD indicate ribonucleotides. b Polyacrylamide gel showing products and efficiency of adapter ligation onto tRNA. Rnl2: T4 RNA Ligase 2. Asterisk (*) indicates 5S and 5.8S ribosomal RNA bands. c Polyacrylamide gel showing products of reverse transcription (cDNA). Rnl2: T4 RNA Ligase 2. d Images of tRNA arrays; each array represents an independent replicate. For the probes spotted at each position see Source Data . e Scatter plot of reads per million derived from QuantM-seq versus array intensities derived from densitometry with a fitted linear trendline. Shaded area represents the 95% confidence interval of the linear trendline. f Scatter plot of northern blot versus array intensities derived from densitometry with a fitted linear trendline. Shaded area represents the 95% confidence interval of the linear trendline. Source data are provided as a Source Data file for ( b – f ).

    Techniques Used: Sequencing, Ligation, Derivative Assay, Northern Blot

    Related Articles

    Functional Assay:

    Article Title: Simple and efficient synthesis of 5? pre-adenylated DNA using thermostable RNA ligase
    Article Snippet: .. This result demonstrates that pre-adenylated DNA, synthesized with MthRnl, is a functional substrate for truncated T4 RNA ligase 2 in the absence of ATP. ..

    Ligation:

    Article Title: Blocking of targeted microRNAs from next-generation sequencing libraries
    Article Snippet: .. This decrease in yield in the 3′ approach is likely due to the leftover ATP from the initial blocking ligation with T4 DNA Ligase inhibiting the truncated T4 RNA Ligase 2 in the subsequent ligation of the adaptor to the 3′ ends of the small RNA pool. .. Although truncated T4 RNA Ligase 2 cannot turnover ATP, ATP can still bind to the remnants of the active site, leading to inhibition of the enzyme (personal communication with NEB).

    Article Title: Simple and efficient synthesis of 5? pre-adenylated DNA using thermostable RNA ligase
    Article Snippet: .. Ten microliter ligation reactions containing 5 pmol of the RNA acceptor, 7 pmol AppDNA17c-NH2 in 10 mM Tris–HCl pH 7.5 buffer, 10 mM MgCl2 , 1 mM DTT and 200 U of truncated T4 RNA ligase 2 were incubated for 2 h at 25°C. .. Reactions were stopped by adding 5 µl formamide loading buffer, heat inactivated at 95°C for 3 min and the products were separated, stained and visualized as described for the DNA adenylation above.

    Article Title: Structure-function analysis of Methanobacterium thermoautotrophicum RNA ligase - engineering a thermostable ATP independent enzyme
    Article Snippet: .. The ligation of pre-adenylated ssDNA or RNA with an acceptor substrate using T4 RNA ligase 1 or truncated T4 RNA ligase 2 (NEB) without ATP were carried out in single-turnover conditions and performed in 10 μl containing 5 pmol of the RNA acceptor, 8 pmol of pre-adenylated donor substrate in 10 mM Tris-HCl pH 7.5 buffer, 10 mM Mg+2 , 1 mM DTT and 200 U (~10 pmol) of truncated T4Rnl2 or 10 U (~50 pmol) of T4Rnl1. .. Standard reactions with a 5’-phosphorylated donor, various acceptors and ATP using T4Rnl1 were performed according to the manufacturer’s protocol (NEB).

    Synthesized:

    Article Title: Simple and efficient synthesis of 5? pre-adenylated DNA using thermostable RNA ligase
    Article Snippet: .. This result demonstrates that pre-adenylated DNA, synthesized with MthRnl, is a functional substrate for truncated T4 RNA ligase 2 in the absence of ATP. ..

    Blocking Assay:

    Article Title: Blocking of targeted microRNAs from next-generation sequencing libraries
    Article Snippet: .. This decrease in yield in the 3′ approach is likely due to the leftover ATP from the initial blocking ligation with T4 DNA Ligase inhibiting the truncated T4 RNA Ligase 2 in the subsequent ligation of the adaptor to the 3′ ends of the small RNA pool. .. Although truncated T4 RNA Ligase 2 cannot turnover ATP, ATP can still bind to the remnants of the active site, leading to inhibition of the enzyme (personal communication with NEB).

    Incubation:

    Article Title: Simple and efficient synthesis of 5? pre-adenylated DNA using thermostable RNA ligase
    Article Snippet: .. Ten microliter ligation reactions containing 5 pmol of the RNA acceptor, 7 pmol AppDNA17c-NH2 in 10 mM Tris–HCl pH 7.5 buffer, 10 mM MgCl2 , 1 mM DTT and 200 U of truncated T4 RNA ligase 2 were incubated for 2 h at 25°C. .. Reactions were stopped by adding 5 µl formamide loading buffer, heat inactivated at 95°C for 3 min and the products were separated, stained and visualized as described for the DNA adenylation above.

    other:

    Article Title: Blocking of targeted microRNAs from next-generation sequencing libraries
    Article Snippet: In the standard protocol (Figure ), a pre-adenylated DNA oligonucleotide adaptor is ligated to the 3′ ends of the pool of small RNA species using truncated T4 RNA Ligase 2.

    Inhibition:

    Article Title: Blocking of targeted microRNAs from next-generation sequencing libraries
    Article Snippet: .. Although truncated T4 RNA Ligase 2 cannot turnover ATP, ATP can still bind to the remnants of the active site, leading to inhibition of the enzyme (personal communication with NEB). .. Thus, a 3′ approach could likely be implemented without unwanted consequences if the reaction components of the blocking ligation were removed via column purification or some other suitable method.

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  • 99
    New England Biolabs t4 rna ligase 2
    Graphical Visualization of the 3′ RACE-Seq Approach, Related to Figure 2 (A) Graphical representation of 3′ RACE-seq library preparation and the oligonucleotides used. First, the 3′ adaptor RA3_15N was joined to the 3′ end of RNA by enzymatic ligation. The adaptor has: (i) 5′ rApp modification for efficient and specific ligation by the truncated <t>T4</t> RNA ligase 2, (ii) delimiter sequence to be used in bioinformatics analyses to exclude RT and PCR artifacts (CTGAC, highlighted in violet), (iii) unique 15N barcode for individual transcript barcoding (highlighted in green), (iv) anchor sequence to pair with the reverse transcription primer (underlined) and (v) dideoxyC on the 3′ end to prevent concatamer formation. The RNA ligated to the adaptor sequence was purified from excess adaptor and reverse transcription was performed with the RT primer, which is compatible with Illumina sequencing and has: (i) sequences to base-pair with the adaptor (underlined), (ii) 6-nucleotide barcode for sample barcoding (highlighted in red), (iii) sequences that base pair with the universal outer primer for nested PCR (blue). Libraries were generated by nested PCR with 2 outer forward primers (F1 and F2) and a single universal reverse primer (uni rev). PCR amplicons of first and second PCRs were purified from excess primers on AmPure beads (Agencourt) before beginning the next step. (B) Flowchart of the bioinformatics approach to 3′ RACE-seq data analysis. The procedure starts at the top. Datasets are shown in rectangles. Software used is depicted in hexagons.
    T4 Rna Ligase 2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 205 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 rna ligase
    Small RNA Northern blot screen reveals a population of tRNA-derived 21–22-nt small RNAs that are 5′-phosphorylated and 3′-hydroxylated. ( A ) Northern blot screen candidate sequences. <t>T4</t> RNA ligase-sensitive small RNAs in bold, except
    T4 Rna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase/product/New England Biolabs
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    Price from $9.99 to $1999.99
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    Image Search Results


    Graphical Visualization of the 3′ RACE-Seq Approach, Related to Figure 2 (A) Graphical representation of 3′ RACE-seq library preparation and the oligonucleotides used. First, the 3′ adaptor RA3_15N was joined to the 3′ end of RNA by enzymatic ligation. The adaptor has: (i) 5′ rApp modification for efficient and specific ligation by the truncated T4 RNA ligase 2, (ii) delimiter sequence to be used in bioinformatics analyses to exclude RT and PCR artifacts (CTGAC, highlighted in violet), (iii) unique 15N barcode for individual transcript barcoding (highlighted in green), (iv) anchor sequence to pair with the reverse transcription primer (underlined) and (v) dideoxyC on the 3′ end to prevent concatamer formation. The RNA ligated to the adaptor sequence was purified from excess adaptor and reverse transcription was performed with the RT primer, which is compatible with Illumina sequencing and has: (i) sequences to base-pair with the adaptor (underlined), (ii) 6-nucleotide barcode for sample barcoding (highlighted in red), (iii) sequences that base pair with the universal outer primer for nested PCR (blue). Libraries were generated by nested PCR with 2 outer forward primers (F1 and F2) and a single universal reverse primer (uni rev). PCR amplicons of first and second PCRs were purified from excess primers on AmPure beads (Agencourt) before beginning the next step. (B) Flowchart of the bioinformatics approach to 3′ RACE-seq data analysis. The procedure starts at the top. Datasets are shown in rectangles. Software used is depicted in hexagons.

    Journal: Cell

    Article Title: Uridylation by TUT4/7 Restricts Retrotransposition of Human LINE-1s

    doi: 10.1016/j.cell.2018.07.022

    Figure Lengend Snippet: Graphical Visualization of the 3′ RACE-Seq Approach, Related to Figure 2 (A) Graphical representation of 3′ RACE-seq library preparation and the oligonucleotides used. First, the 3′ adaptor RA3_15N was joined to the 3′ end of RNA by enzymatic ligation. The adaptor has: (i) 5′ rApp modification for efficient and specific ligation by the truncated T4 RNA ligase 2, (ii) delimiter sequence to be used in bioinformatics analyses to exclude RT and PCR artifacts (CTGAC, highlighted in violet), (iii) unique 15N barcode for individual transcript barcoding (highlighted in green), (iv) anchor sequence to pair with the reverse transcription primer (underlined) and (v) dideoxyC on the 3′ end to prevent concatamer formation. The RNA ligated to the adaptor sequence was purified from excess adaptor and reverse transcription was performed with the RT primer, which is compatible with Illumina sequencing and has: (i) sequences to base-pair with the adaptor (underlined), (ii) 6-nucleotide barcode for sample barcoding (highlighted in red), (iii) sequences that base pair with the universal outer primer for nested PCR (blue). Libraries were generated by nested PCR with 2 outer forward primers (F1 and F2) and a single universal reverse primer (uni rev). PCR amplicons of first and second PCRs were purified from excess primers on AmPure beads (Agencourt) before beginning the next step. (B) Flowchart of the bioinformatics approach to 3′ RACE-seq data analysis. The procedure starts at the top. Datasets are shown in rectangles. Software used is depicted in hexagons.

    Article Snippet: The reactions were carried out in 20 μL with 1x T4 RNA ligase 2 truncated buffer (NEB) supplemented with PEG-8000 at 10% final concentration, 0.25 U/μl RiboLock inhibitor (Thermo Fisher Scientific), 3 pmol of the 5′ FAM-labeled 44-mer oligonucleotide RNA44 (Future Synthesis) and 300 U T4 RNA ligase 2 truncated (NEB) for 18h at 18°C.

    Techniques: Ligation, Modification, Sequencing, Polymerase Chain Reaction, Purification, Nested PCR, Generated, Software

    Small RNA Northern blot screen reveals a population of tRNA-derived 21–22-nt small RNAs that are 5′-phosphorylated and 3′-hydroxylated. ( A ) Northern blot screen candidate sequences. T4 RNA ligase-sensitive small RNAs in bold, except

    Journal: RNA

    Article Title: Human tRNA-derived small RNAs in the global regulation of RNA silencing

    doi: 10.1261/rna.2000810

    Figure Lengend Snippet: Small RNA Northern blot screen reveals a population of tRNA-derived 21–22-nt small RNAs that are 5′-phosphorylated and 3′-hydroxylated. ( A ) Northern blot screen candidate sequences. T4 RNA ligase-sensitive small RNAs in bold, except

    Article Snippet: Amounts of enzymes used: 15 units (U) of T4 PNK, 3′ phophatase ± (NEB M0201/m0236); 8 U of Tobacco Acid Pyrophosphatase (Epicentre Biotechnologies); 3 U of Terminator Exonuclease (Epicentre Biotechnologies); 4 U polyA polymerase (PAP; Ambion); and 15 U T4 RNA ligase (NEB).

    Techniques: Northern Blot, Derivative Assay

    The RNA-ligase-mediated 5′-RACE procedure to clone the 5′ terminus of a viral RNA genome. Viral RNA is converted to cDNA with random primers by reverse transcription. A phosphorylated synthetic oligodeoxynucleotide adapter is ligated to the 3′ end of cDNA by using T4 RNA ligase, and then two rounds of PCR amplification are performed. The first-round PCR is done with the adapter primer (AP-1), complementary to the 3′ end of the adapter, and the viral-specific primer 1 (VS-1). The second-round nested PCR is done with the other adapter primer (AP-2), complementary to the 5′ portion of the adapter, and the viral-specific primer 2 (VS-2). The PCR products are subjected to cloning and then sequencing. The RNA molecule is represented as a wavy line, cDNA molecules are straight lines, adapters are thick lines, and primers are short arrows.

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

    Article Title: Cloning and characterization of the extreme 5?-terminal sequences of the RNA genomes of GB virus C/hepatitis G virus

    doi:

    Figure Lengend Snippet: The RNA-ligase-mediated 5′-RACE procedure to clone the 5′ terminus of a viral RNA genome. Viral RNA is converted to cDNA with random primers by reverse transcription. A phosphorylated synthetic oligodeoxynucleotide adapter is ligated to the 3′ end of cDNA by using T4 RNA ligase, and then two rounds of PCR amplification are performed. The first-round PCR is done with the adapter primer (AP-1), complementary to the 3′ end of the adapter, and the viral-specific primer 1 (VS-1). The second-round nested PCR is done with the other adapter primer (AP-2), complementary to the 5′ portion of the adapter, and the viral-specific primer 2 (VS-2). The PCR products are subjected to cloning and then sequencing. The RNA molecule is represented as a wavy line, cDNA molecules are straight lines, adapters are thick lines, and primers are short arrows.

    Article Snippet: The ligation solution contained 5 pg of the synthetic oligonucleotide adapter, 50 mM Tris·HCl (pH 7.8), 10 mM MgCl2 , 1 mM 2-mercaptoethanol, 1 mM ATP, 20 units of human placenta ribonuclease inhibitor (RNasin, Promega), and 20 units of T4 RNA ligase (New England BioLabs).

    Techniques: Polymerase Chain Reaction, Amplification, Nested PCR, Clone Assay, Sequencing

    Main cleavage sites in the 25S rRNA are located in loop regions. ( A ) Northern hybridizations of total yeast RNA extracted from wild-type W303 cells treated with different concentrations of H 2 O 2 . Hybridizations with probes against 25S (probe 007, position +40, lanes 1–6; probe W234, position +344, lanes 7–12; probe W236, position +600, lanes 13–18; probe W238, position +843, lanes 19–24; probe W239, position +2168, lanes 25–30 and probe W240, position +3323, lanes 31–36). Asterisks above the arrows indicate the products that were further analysed. Arrow marked with a hatch shows a band matching the potential 3′ product of the major cleavage, 5′ product is marked with one asterisk. ( B–C ) Primer extension analysis for two main cleavage sites in the 25S rRNA in W303 cells treated with 1 mM H 2 O 2 (A) and in 16-day old chronologically aged rho0 W303 cells (B). Primer extensions were performed using primers W235 for sites around positions +400 and +470 and W237 for sites around position +600 relative to the 5′ end of the mature 25S. DNA sequencing on a PCR product encompassing the 5′ end of the 25S from +40 to +701, using the same primers was run in parallel on 6% sequencing polyacrylamide gels (lanes 1–4). The sequences with primer extension stops are shown on the right. Secondary structures of the regions in the vicinity of the cleavages, indicated by arrowheads and shown beside corresponding primer extension reactions, were adapted from the website http://rna.icmb.utexas.edu/ . ( D–E ) 3′ ends of cleaved-off products for the major cleavage at positions +610–611 were mapped by 3′ RACE. (D) PCR reactions on cDNA prepared using total RNA from untreated control (lane 1, C) and cells treated with 1 mM H 2 O 2 (lane 2). To generate cDNA, total RNA that had been ligated to an ‘anchor’ oligonucleotide (W242) with T4 RNA ligase, was reverse transcribed using a primer specific for the anchor (W243). This was followed by PCR reaction using the same 3′ primer and the 5′ primer starting at position +50 in the 25S rRNA (W241). Arrows indicate products corresponding to fragments cleaved at +398–404 (lower) and +610–611 (upper). PCR fragments were cloned into pGEM-Teasy and sequenced. (E) Sequences obtained by the 3′ RACE analysis for fragments cleaved at site +398–403 (19 independent clones) and site +610–611 (10 independent clones). The corresponding regions of the 25S with cleavage sites mapped by primer extension and indicated with empty arrowheads are shown above in grey. Figures in parentheses show the number of identical clones. ( F ) Mapping 3′ ends of two major cleavages sites using RNase H cleavage on total RNA extracted from wild-type, rrp41-1 and ski7Δ cells treated with 1mM H 2 O 2 (lanes, 2–4) and from wild-type untreated control (lane 1, C). RNase H treatment was performed on RNA samples annealed to DNA oligonucleotides W244 and W263 complementary to positions +271 and +510, respectively. Samples were separated on a 8% acrylamide gel and hybridized with probe W234 (F-I) and probe W264 (F-II) to detect 3′ ends of fragments cleaved at +398–403 (F-I) and at +610–611 (F-II), respectively. Arrows show more defined 3′ ends of products cleaved at +610–611 for all strains and at +398–403 in the mutants; vertical bar in F-I indicates heterogenous 3′ ends of products cleaved at +398–403 in wild-type cells.

    Journal: Nucleic Acids Research

    Article Title: Apoptotic signals induce specific degradation of ribosomal RNA in yeast

    doi: 10.1093/nar/gkm1100

    Figure Lengend Snippet: Main cleavage sites in the 25S rRNA are located in loop regions. ( A ) Northern hybridizations of total yeast RNA extracted from wild-type W303 cells treated with different concentrations of H 2 O 2 . Hybridizations with probes against 25S (probe 007, position +40, lanes 1–6; probe W234, position +344, lanes 7–12; probe W236, position +600, lanes 13–18; probe W238, position +843, lanes 19–24; probe W239, position +2168, lanes 25–30 and probe W240, position +3323, lanes 31–36). Asterisks above the arrows indicate the products that were further analysed. Arrow marked with a hatch shows a band matching the potential 3′ product of the major cleavage, 5′ product is marked with one asterisk. ( B–C ) Primer extension analysis for two main cleavage sites in the 25S rRNA in W303 cells treated with 1 mM H 2 O 2 (A) and in 16-day old chronologically aged rho0 W303 cells (B). Primer extensions were performed using primers W235 for sites around positions +400 and +470 and W237 for sites around position +600 relative to the 5′ end of the mature 25S. DNA sequencing on a PCR product encompassing the 5′ end of the 25S from +40 to +701, using the same primers was run in parallel on 6% sequencing polyacrylamide gels (lanes 1–4). The sequences with primer extension stops are shown on the right. Secondary structures of the regions in the vicinity of the cleavages, indicated by arrowheads and shown beside corresponding primer extension reactions, were adapted from the website http://rna.icmb.utexas.edu/ . ( D–E ) 3′ ends of cleaved-off products for the major cleavage at positions +610–611 were mapped by 3′ RACE. (D) PCR reactions on cDNA prepared using total RNA from untreated control (lane 1, C) and cells treated with 1 mM H 2 O 2 (lane 2). To generate cDNA, total RNA that had been ligated to an ‘anchor’ oligonucleotide (W242) with T4 RNA ligase, was reverse transcribed using a primer specific for the anchor (W243). This was followed by PCR reaction using the same 3′ primer and the 5′ primer starting at position +50 in the 25S rRNA (W241). Arrows indicate products corresponding to fragments cleaved at +398–404 (lower) and +610–611 (upper). PCR fragments were cloned into pGEM-Teasy and sequenced. (E) Sequences obtained by the 3′ RACE analysis for fragments cleaved at site +398–403 (19 independent clones) and site +610–611 (10 independent clones). The corresponding regions of the 25S with cleavage sites mapped by primer extension and indicated with empty arrowheads are shown above in grey. Figures in parentheses show the number of identical clones. ( F ) Mapping 3′ ends of two major cleavages sites using RNase H cleavage on total RNA extracted from wild-type, rrp41-1 and ski7Δ cells treated with 1mM H 2 O 2 (lanes, 2–4) and from wild-type untreated control (lane 1, C). RNase H treatment was performed on RNA samples annealed to DNA oligonucleotides W244 and W263 complementary to positions +271 and +510, respectively. Samples were separated on a 8% acrylamide gel and hybridized with probe W234 (F-I) and probe W264 (F-II) to detect 3′ ends of fragments cleaved at +398–403 (F-I) and at +610–611 (F-II), respectively. Arrows show more defined 3′ ends of products cleaved at +610–611 for all strains and at +398–403 in the mutants; vertical bar in F-I indicates heterogenous 3′ ends of products cleaved at +398–403 in wild-type cells.

    Article Snippet: To this end, DNA ‘anchor’ oligonucleotide (W242) was ligated with T4 RNA ligase to total RNA from untreated and treated W303 cells to prepare cDNA using a primer specific for the anchor (W243).

    Techniques: Northern Blot, DNA Sequencing, Polymerase Chain Reaction, Sequencing, Clone Assay, Acrylamide Gel Assay