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 927 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase 2 - by Bioz Stars, 2020-09
    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 "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

    3) 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

    4) Product Images from "Decreasing miRNA sequencing bias using a single adapter and circularization approach"

    Article Title: Decreasing miRNA sequencing bias using a single adapter and circularization approach

    Journal: Genome Biology

    doi: 10.1186/s13059-018-1488-z

    Bias in miRNA detection using various small-RNA library preparation kits. For each kit, sequencing libraries were prepared from the miRXplore™ pool and sequenced; the sequence data were then used to calculate fold-deviations from the equimolar input and plotted as log 2 values. Densities of miRNAs within a two-fold deviation from the expected values (between vertical lines ]. Under-represented, over-represented, and accurately quantified percentages of miRNAs are shown in red font . Results for two-adapter schemes are a TruSeq® Small RNA, b NEBNext®, and c QIAseq. d NEXTFlex™, a scheme using two adapters with randomized sequences. e SMARTer, which uses template switching. f RealSeq®-AC, which uses a single-adapter and circularization (* p value vs other kits
    Figure Legend Snippet: Bias in miRNA detection using various small-RNA library preparation kits. For each kit, sequencing libraries were prepared from the miRXplore™ pool and sequenced; the sequence data were then used to calculate fold-deviations from the equimolar input and plotted as log 2 values. Densities of miRNAs within a two-fold deviation from the expected values (between vertical lines ]. Under-represented, over-represented, and accurately quantified percentages of miRNAs are shown in red font . Results for two-adapter schemes are a TruSeq® Small RNA, b NEBNext®, and c QIAseq. d NEXTFlex™, a scheme using two adapters with randomized sequences. e SMARTer, which uses template switching. f RealSeq®-AC, which uses a single-adapter and circularization (* p value vs other kits

    Techniques Used: Sequencing

    Correlation of miRNA reads between libraries created with 100 ng, 10 ng, or 1 ng inputs of Human Reference RNA (Agilent). Raw reads mapping to miRNAs were used to calculate the Pearson correlation between libraries
    Figure Legend Snippet: Correlation of miRNA reads between libraries created with 100 ng, 10 ng, or 1 ng inputs of Human Reference RNA (Agilent). Raw reads mapping to miRNAs were used to calculate the Pearson correlation between libraries

    Techniques Used:

    5) 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

    6) 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

    7) Product Images from "Multiple ribonuclease A family members cleave transfer RNAs in response to stress"

    Article Title: Multiple ribonuclease A family members cleave transfer RNAs in response to stress

    Journal: bioRxiv

    doi: 10.1101/811174

    Validation of CCA-specific ligation methods. (A) Schema for three CCA-specific ligation methods. Dnl: T4 DNA ligase, Rnl2: T4 RNA ligase 2, bio: biotin. (B-C) The method using double-strand oligo and RNA ligase 2 has the best ligation efficiency. (B) SYBR Gold staining and (C) Northern blotting for CCA-specific ligation products. The blue arrowheads indicate the bands for pre-tRNAs.
    Figure Legend Snippet: Validation of CCA-specific ligation methods. (A) Schema for three CCA-specific ligation methods. Dnl: T4 DNA ligase, Rnl2: T4 RNA ligase 2, bio: biotin. (B-C) The method using double-strand oligo and RNA ligase 2 has the best ligation efficiency. (B) SYBR Gold staining and (C) Northern blotting for CCA-specific ligation products. The blue arrowheads indicate the bands for pre-tRNAs.

    Techniques Used: Ligation, Staining, Northern Blot

    8) 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

    9) 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

    10) Product Images from "Multiple ribonuclease A family members cleave transfer RNAs in response to stress"

    Article Title: Multiple ribonuclease A family members cleave transfer RNAs in response to stress

    Journal: bioRxiv

    doi: 10.1101/811174

    Validation of CCA-specific ligation methods. (A) Schema for three CCA-specific ligation methods. Dnl: T4 DNA ligase, Rnl2: T4 RNA ligase 2, bio: biotin. (B-C) The method using double-strand oligo and RNA ligase 2 has the best ligation efficiency. (B) SYBR Gold staining and (C) Northern blotting for CCA-specific ligation products. The blue arrowheads indicate the bands for pre-tRNAs.
    Figure Legend Snippet: Validation of CCA-specific ligation methods. (A) Schema for three CCA-specific ligation methods. Dnl: T4 DNA ligase, Rnl2: T4 RNA ligase 2, bio: biotin. (B-C) The method using double-strand oligo and RNA ligase 2 has the best ligation efficiency. (B) SYBR Gold staining and (C) Northern blotting for CCA-specific ligation products. The blue arrowheads indicate the bands for pre-tRNAs.

    Techniques Used: Ligation, Staining, Northern Blot

    11) Product Images from "Decreasing miRNA sequencing bias using a single adapter and circularization approach"

    Article Title: Decreasing miRNA sequencing bias using a single adapter and circularization approach

    Journal: Genome Biology

    doi: 10.1186/s13059-018-1488-z

    Bias in miRNA detection using various small-RNA library preparation kits. For each kit, sequencing libraries were prepared from the miRXplore™ pool and sequenced; the sequence data were then used to calculate fold-deviations from the equimolar input and plotted as log 2 values. Densities of miRNAs within a two-fold deviation from the expected values (between vertical lines ]. Under-represented, over-represented, and accurately quantified percentages of miRNAs are shown in red font . Results for two-adapter schemes are a TruSeq® Small RNA, b NEBNext®, and c QIAseq. d NEXTFlex™, a scheme using two adapters with randomized sequences. e SMARTer, which uses template switching. f RealSeq®-AC, which uses a single-adapter and circularization (* p value vs other kits
    Figure Legend Snippet: Bias in miRNA detection using various small-RNA library preparation kits. For each kit, sequencing libraries were prepared from the miRXplore™ pool and sequenced; the sequence data were then used to calculate fold-deviations from the equimolar input and plotted as log 2 values. Densities of miRNAs within a two-fold deviation from the expected values (between vertical lines ]. Under-represented, over-represented, and accurately quantified percentages of miRNAs are shown in red font . Results for two-adapter schemes are a TruSeq® Small RNA, b NEBNext®, and c QIAseq. d NEXTFlex™, a scheme using two adapters with randomized sequences. e SMARTer, which uses template switching. f RealSeq®-AC, which uses a single-adapter and circularization (* p value vs other kits

    Techniques Used: Sequencing

    Correlation of miRNA reads between libraries created with 100 ng, 10 ng, or 1 ng inputs of Human Reference RNA (Agilent). Raw reads mapping to miRNAs were used to calculate the Pearson correlation between libraries
    Figure Legend Snippet: Correlation of miRNA reads between libraries created with 100 ng, 10 ng, or 1 ng inputs of Human Reference RNA (Agilent). Raw reads mapping to miRNAs were used to calculate the Pearson correlation between libraries

    Techniques Used:

    12) 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

    13) 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

    14) Product Images from "Sensitive and specific miRNA detection method using SplintR Ligase"

    Article Title: Sensitive and specific miRNA detection method using SplintR Ligase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw399

    Comparison of T4 DNA Ligase, T4 RNA Ligase 2 and SplintR Ligase for their ability to join different length DNA probes hybridized to miR-122. ( A ) Pairs of DNA oligonucleotides complementary to miR-122 were used to determine the minimum DNA:RNA overlap required for ligation. The miR-122 sequence (red) is complementary to probe A (green) and probe B (black). Probe A and probe B are designed to scan the miRNA sequence in two base increments. Probe A has a 5′ FAM label and probe B has a 5′ phosphate that allows ligation. Each pair of DNA probes is named by the number of nucleotides (nt) that are complementary to the 22 nt in miR-122, for example probe A8 has 8 nt and B14 has 14 nt complementary to miR-122. ( B ) The ligation of FAM labeled probe A to probe B is visualized by a fluorescent scan after electrophoresis on a 15% TBE urea acrylamide gel. Probe B is not observed because only probe A has a FAM label. Since there is more probe A than miR-122 each reaction has both ligated and unligated probe. Lane M, at the left of the gel, denotes the size in nucleotides of the FAM labeled ssDNA marker. The specific components and reaction conditions are described in Materials and Methods. ( C ) The ligation of DNA probes hybridized to miR-122 by SplintR Ligase, T4 DNA Ligase or T4 RNA Ligase 2 (T4 RL2) (indicated on the left) at different incubation times was measured by capillary electrophoresis (CE) fragment analysis. The traces are shown for 5, 30 and 60 minute ligations of probes gA10 and gB12 hybridized to miR-122. Position of the 32 nt substrate (32(S)) and the 65 nt ligated product (65(P)) are indicated on the top of the panels. Reaction conditions are listed in Materials and Methods. ( D ) Ligation time course for the three enzymes, SpllintR (red), T4 DNA ligase (black) and T4 RNA ligase 2 (green) was measured by CE. The percent of ligation is indicated on the left of the graph.
    Figure Legend Snippet: Comparison of T4 DNA Ligase, T4 RNA Ligase 2 and SplintR Ligase for their ability to join different length DNA probes hybridized to miR-122. ( A ) Pairs of DNA oligonucleotides complementary to miR-122 were used to determine the minimum DNA:RNA overlap required for ligation. The miR-122 sequence (red) is complementary to probe A (green) and probe B (black). Probe A and probe B are designed to scan the miRNA sequence in two base increments. Probe A has a 5′ FAM label and probe B has a 5′ phosphate that allows ligation. Each pair of DNA probes is named by the number of nucleotides (nt) that are complementary to the 22 nt in miR-122, for example probe A8 has 8 nt and B14 has 14 nt complementary to miR-122. ( B ) The ligation of FAM labeled probe A to probe B is visualized by a fluorescent scan after electrophoresis on a 15% TBE urea acrylamide gel. Probe B is not observed because only probe A has a FAM label. Since there is more probe A than miR-122 each reaction has both ligated and unligated probe. Lane M, at the left of the gel, denotes the size in nucleotides of the FAM labeled ssDNA marker. The specific components and reaction conditions are described in Materials and Methods. ( C ) The ligation of DNA probes hybridized to miR-122 by SplintR Ligase, T4 DNA Ligase or T4 RNA Ligase 2 (T4 RL2) (indicated on the left) at different incubation times was measured by capillary electrophoresis (CE) fragment analysis. The traces are shown for 5, 30 and 60 minute ligations of probes gA10 and gB12 hybridized to miR-122. Position of the 32 nt substrate (32(S)) and the 65 nt ligated product (65(P)) are indicated on the top of the panels. Reaction conditions are listed in Materials and Methods. ( D ) Ligation time course for the three enzymes, SpllintR (red), T4 DNA ligase (black) and T4 RNA ligase 2 (green) was measured by CE. The percent of ligation is indicated on the left of the graph.

    Techniques Used: Ligation, Sequencing, Labeling, Electrophoresis, Acrylamide Gel Assay, Marker, Incubation

    15) Product Images from "Decreasing miRNA sequencing bias using a single adapter and circularization approach"

    Article Title: Decreasing miRNA sequencing bias using a single adapter and circularization approach

    Journal: Genome Biology

    doi: 10.1186/s13059-018-1488-z

    Bias in miRNA detection using various small-RNA library preparation kits. For each kit, sequencing libraries were prepared from the miRXplore™ pool and sequenced; the sequence data were then used to calculate fold-deviations from the equimolar input and plotted as log 2 values. Densities of miRNAs within a two-fold deviation from the expected values (between vertical lines ) are considered unbiased according to [ 8 ]. Under-represented, over-represented, and accurately quantified percentages of miRNAs are shown in red font . Results for two-adapter schemes are a TruSeq® Small RNA, b NEBNext®, and c QIAseq. d NEXTFlex™, a scheme using two adapters with randomized sequences. e SMARTer, which uses template switching. f RealSeq®-AC, which uses a single-adapter and circularization (* p value vs other kits
    Figure Legend Snippet: Bias in miRNA detection using various small-RNA library preparation kits. For each kit, sequencing libraries were prepared from the miRXplore™ pool and sequenced; the sequence data were then used to calculate fold-deviations from the equimolar input and plotted as log 2 values. Densities of miRNAs within a two-fold deviation from the expected values (between vertical lines ) are considered unbiased according to [ 8 ]. Under-represented, over-represented, and accurately quantified percentages of miRNAs are shown in red font . Results for two-adapter schemes are a TruSeq® Small RNA, b NEBNext®, and c QIAseq. d NEXTFlex™, a scheme using two adapters with randomized sequences. e SMARTer, which uses template switching. f RealSeq®-AC, which uses a single-adapter and circularization (* p value vs other kits

    Techniques Used: Sequencing

    Differential quantification of brain samples between different small RNA library preparation kits. Data obtained with either a TruSeq®, b NEBNext®, c NEXTFlex™, d QIAseq, or e SMARTer kits were compared with data obtained with RealSeq®-AC to obtain differential quantification (log 2 ) values for 276 high-confidence miRNAs. These values were plotted against the accuracy of detection of that miRNA when profiling the equimolar pool of synthetic miRNAs (Fig. 2 a–c). f–j The reverse comparison, with the differential quantification of RealSeq®-AC versus each of the other kits plotted against the accuracy of RealSeq®-AC when quantifying the synthetic pool of miRNAs. FN false negative, FP false positive. See Methods for more details
    Figure Legend Snippet: Differential quantification of brain samples between different small RNA library preparation kits. Data obtained with either a TruSeq®, b NEBNext®, c NEXTFlex™, d QIAseq, or e SMARTer kits were compared with data obtained with RealSeq®-AC to obtain differential quantification (log 2 ) values for 276 high-confidence miRNAs. These values were plotted against the accuracy of detection of that miRNA when profiling the equimolar pool of synthetic miRNAs (Fig. 2 a–c). f–j The reverse comparison, with the differential quantification of RealSeq®-AC versus each of the other kits plotted against the accuracy of RealSeq®-AC when quantifying the synthetic pool of miRNAs. FN false negative, FP false positive. See Methods for more details

    Techniques Used:

    Correlation of miRNA reads between libraries created with 100 ng, 10 ng, or 1 ng inputs of Human Reference RNA (Agilent). Raw reads mapping to miRNAs were used to calculate the Pearson correlation between libraries
    Figure Legend Snippet: Correlation of miRNA reads between libraries created with 100 ng, 10 ng, or 1 ng inputs of Human Reference RNA (Agilent). Raw reads mapping to miRNAs were used to calculate the Pearson correlation between libraries

    Techniques Used:

    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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    New England Biolabs t4 rnl2 reaction buffer
    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.
    T4 Rnl2 Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkx073

    Figure Lengend 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.

    Article Snippet: The ligation reaction was performed in 20 μl containing 1× T4 Rnl2 reaction buffer (NEB), 12.5% (w/v) polyethylene glycol (PEG) 8000 or PEG 4000 and 5 units of T4 RNA Ligase 2 (NEB).

    Techniques: Ligation, Transformation Assay

    Detection of sPom121 mRNA and protein expression in human cells. ( A ) Schematic of two putative Pom121 isoforms expressed in humans and confirmed by 5′ rapid amplification of cDNA ends (RACE). ( Top ) Gray boxes indicate 5′ UTR-encoding exons, blue boxes indicate TM domain-encoding exon, and black boxes indicate Pom121-encoding exons. ( Bottom ) The TM domain, nuclear localization signal (NLS), and phenylalanine/glycine (FG) domain are indicated (blue, red, and green, respectively). ( B ) Schematic showing the annotated 5′ end of the Pom121 gene (shown at the top , “Pom121 gene”) compared with that of sPom121 (“5′ RACE sPom121 mRNA”) and Pom121 (“5′ RACE Pom121 mRNA”), identified here by 5′ RACE. ( C ) Histone H3 Lys4 trimethylation (H3K4me3) ( top ) and RNA sequencing (RNA-seq) ( bottom ) results from HeLa-C cells. Red arrows are used to indicate active transcriptional start sites ( top ), sPom121-specific exons ( bottom left ), or the TM-coding exon of Pom121 ( bottom right ). ( D ) sPom121 and Pom121 expression in various tissues. Quantitative PCR (qPCR) analysis of sPom121 (red bars) and Pom121 (blue bars) mRNA levels in multiple tissue types relative to actin. Results are plotted such that the tissue with the lowest sPom121 mRNA expression is at the left , while the tissue expressing the highest levels of sPom121 is shown at the right . Different primers were used to analyze sPom121 and Pom121 cDNA levels, and thus a comparison of sPom121 and Pom121 levels in each tissue cannot be made from these data. ( E ) Western blot to detect sPom121. Soluble (lanes 1 , 2 ) and insoluble (lanes 3 , 4 ) lysates were electrophoresed and Western blotted, and proteins were detected with a Pom121 antibody ( top panels) or tubulin ( bottom panels). (Lanes 2 , 4 ) Samples that had been treated with Pom121 siRNA are included to identify which bands correspond to Pom121 protein. Pom121 blots were exposed for 30 sec ( left blot) or 10 sec ( right blot).

    Journal: Genes & Development

    Article Title: Evolution of a transcriptional regulator from a transmembrane nucleoporin

    doi: 10.1101/gad.280941.116

    Figure Lengend Snippet: Detection of sPom121 mRNA and protein expression in human cells. ( A ) Schematic of two putative Pom121 isoforms expressed in humans and confirmed by 5′ rapid amplification of cDNA ends (RACE). ( Top ) Gray boxes indicate 5′ UTR-encoding exons, blue boxes indicate TM domain-encoding exon, and black boxes indicate Pom121-encoding exons. ( Bottom ) The TM domain, nuclear localization signal (NLS), and phenylalanine/glycine (FG) domain are indicated (blue, red, and green, respectively). ( B ) Schematic showing the annotated 5′ end of the Pom121 gene (shown at the top , “Pom121 gene”) compared with that of sPom121 (“5′ RACE sPom121 mRNA”) and Pom121 (“5′ RACE Pom121 mRNA”), identified here by 5′ RACE. ( C ) Histone H3 Lys4 trimethylation (H3K4me3) ( top ) and RNA sequencing (RNA-seq) ( bottom ) results from HeLa-C cells. Red arrows are used to indicate active transcriptional start sites ( top ), sPom121-specific exons ( bottom left ), or the TM-coding exon of Pom121 ( bottom right ). ( D ) sPom121 and Pom121 expression in various tissues. Quantitative PCR (qPCR) analysis of sPom121 (red bars) and Pom121 (blue bars) mRNA levels in multiple tissue types relative to actin. Results are plotted such that the tissue with the lowest sPom121 mRNA expression is at the left , while the tissue expressing the highest levels of sPom121 is shown at the right . Different primers were used to analyze sPom121 and Pom121 cDNA levels, and thus a comparison of sPom121 and Pom121 levels in each tissue cannot be made from these data. ( E ) Western blot to detect sPom121. Soluble (lanes 1 , 2 ) and insoluble (lanes 3 , 4 ) lysates were electrophoresed and Western blotted, and proteins were detected with a Pom121 antibody ( top panels) or tubulin ( bottom panels). (Lanes 2 , 4 ) Samples that had been treated with Pom121 siRNA are included to identify which bands correspond to Pom121 protein. Pom121 blots were exposed for 30 sec ( left blot) or 10 sec ( right blot).

    Article Snippet: Next, an anchor primer was ligated to the 3′ end of the Pom121 cDNA products (4 µL of Pom121 cDNA, 2 µL of phosphorylated anchor primer, 2 µL of RNA ligase buffer, 8 µL of 50% PEG 8000, 1 µL of 10 mM ATP, 1 µL of 0.1 M DTT, 1 µL of SS RNA ligase 1 [New England Biolabs]) overnight at 25°C.

    Techniques: Expressing, Rapid Amplification of cDNA Ends, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Western Blot, Size-exclusion Chromatography

    TSS-EMOTE flowchart. The TSS-EMOTE assay consists of a wet-lab library preparation (panels a to g ) and in silico analyses (panel H to N). An asterisk continually marks the original 5’-base of tri-phosphorylated RNA (thin red line). a Total RNA is purified, and digested with XRN1 5’-exonuclease, which removes the vast majority of 5’ mono-phosphorylated RNA from the sample (including 16S and 23S rRNA). b and c The XRN1 treated RNA is mixed with large excess of a synthetic RNA oligo (Rp6, shown in blue), and split into two pools. Both pools receive T4 RNA ligase, but only the “+RppH” pool is co-treated with RppH, an enzyme that converts 5’ tri-phosphorylated ends to mono-phosphorylated ends, thus allowing the ligase to use them as substrates. d and e After the ligation reaction, a semi-random primer is used to reverse-transcribe the RNA and simultaneously add a 2.0 Illumina adapter (black “B”). This results in cDNA with a 2.0 Illumina adaptor (for reverse reads in paired-end sequencing) at the 5’-end and if the template RNA was ligated to an Rp6 oligo, then the cDNA will also have a complementary sequence to Rp6 at the 3’-end (cRp6). f PCR is used to specifically amplify cDNAs that carry the 2.0 Illumina adaptor and cRp6 sequences. This step moreover adds a 1.0 Illumina adaptor (for forward reads in paired-end sequencing) and a sample-specific 4-base EMOTE barcode (blue line and “XXX”, respectively) to index the molecules (different barcodes for the -RppH and + RppH pools). The barcode of the -RppH pool will designate molecules where the XRN1 treatments has been incomplete, and this information is incorporated into the in silico analysis (see below). g The barcoded DNA from various samples (and pools) can be mixed, and loaded directly into an Illumina HiSeq machine. Millions of 50 nt sequences are obtained, each of which will span the EMOTE barcode, both known and random sections of the Rp6 oligo (see Methods ), and it will reveal the first 20 nt of the native 5’-end of the ligated RNA molecule. These 20 nt are sufficient to map the vast majority of 5’-ends to a unique position on the small genomes of the bacteria in this study. However, longer Illumina reads (and thus longer mapping sequences) can be used if the TSSs are in repeated regions or if large-genome organisms, such as humans, are being examined. h The in silico pipeline input consists of stranded RNA-seq reads for one or multiple biological replicates in FASTQ format. Each replicate includes a FASTQ for the -RppH pool and another for the + RppH pool. i The FASTQ files go through EMOTE-conv software [ 51 ] that parses the reads, aligns them to the genome, and perform the quantification. Thus, for each genomic position we obtain the number of reads whose first nucleotide align at this genomic position, and on which strand it maps. The counts are further corrected for PCR biases by looking at the unique molecular identifiers (UMIs) sequences available in the unaligned part of the EMOTE read. j Quantification counts obtained for + RppH and -RppH pools are compared through a beta-binomial model that tests whether the identified 5’ ends in the + RppH pool is significantly enriched over the identified 5’ ends in the -RppH pool at a given position. The process results in a p-value that reflects our confidence in the genomic position to be enriched in the + RppH pool of the biological replicate. k The p-values of all the biological replicates are combined into a single p-value with Fisher’s method. l and m To correct the p-values for multiple testing across all genomic positions, the false discovery rate (FDR) is evaluated and only those with a FDR ≤ 0.01 are considered to be TSSs. Note also that for the FDR is only calculated for genomic positions with at least 5 detected ligation-events in at least one of the + RppH pools (UMI ≥ 5). n The TSSs then enter an annotation process that retrieve their surrounding sequence and downstream ORFs. TSSs separated by less than 5 bp are clustered together. Finally, to draw a global picture of operon structures, an independent detection of transcription terminators is operated with the software TransTermHP [ 39 ]. o Sequence of the RNA oligo Rp6 and a typical Illumina sequencing read from a TSS-EMOTE experiment. The Recognition Sequence serves as priming site for the PCR in panel F. UMI: The randomly incorporated nucleotides in the Rp6 oligo that serves to whether Illumina reads with identical Mapping Sequences originate from separate ligation events. CS: Control Sequence. EB: EMOTE barcode to index the Illumina reads. An asterisk indicates the 5’ nucleotide of the original RNA molecule

    Journal: BMC Genomics

    Article Title: TSS-EMOTE, a refined protocol for a more complete and less biased global mapping of transcription start sites in bacterial pathogens

    doi: 10.1186/s12864-016-3211-3

    Figure Lengend Snippet: TSS-EMOTE flowchart. The TSS-EMOTE assay consists of a wet-lab library preparation (panels a to g ) and in silico analyses (panel H to N). An asterisk continually marks the original 5’-base of tri-phosphorylated RNA (thin red line). a Total RNA is purified, and digested with XRN1 5’-exonuclease, which removes the vast majority of 5’ mono-phosphorylated RNA from the sample (including 16S and 23S rRNA). b and c The XRN1 treated RNA is mixed with large excess of a synthetic RNA oligo (Rp6, shown in blue), and split into two pools. Both pools receive T4 RNA ligase, but only the “+RppH” pool is co-treated with RppH, an enzyme that converts 5’ tri-phosphorylated ends to mono-phosphorylated ends, thus allowing the ligase to use them as substrates. d and e After the ligation reaction, a semi-random primer is used to reverse-transcribe the RNA and simultaneously add a 2.0 Illumina adapter (black “B”). This results in cDNA with a 2.0 Illumina adaptor (for reverse reads in paired-end sequencing) at the 5’-end and if the template RNA was ligated to an Rp6 oligo, then the cDNA will also have a complementary sequence to Rp6 at the 3’-end (cRp6). f PCR is used to specifically amplify cDNAs that carry the 2.0 Illumina adaptor and cRp6 sequences. This step moreover adds a 1.0 Illumina adaptor (for forward reads in paired-end sequencing) and a sample-specific 4-base EMOTE barcode (blue line and “XXX”, respectively) to index the molecules (different barcodes for the -RppH and + RppH pools). The barcode of the -RppH pool will designate molecules where the XRN1 treatments has been incomplete, and this information is incorporated into the in silico analysis (see below). g The barcoded DNA from various samples (and pools) can be mixed, and loaded directly into an Illumina HiSeq machine. Millions of 50 nt sequences are obtained, each of which will span the EMOTE barcode, both known and random sections of the Rp6 oligo (see Methods ), and it will reveal the first 20 nt of the native 5’-end of the ligated RNA molecule. These 20 nt are sufficient to map the vast majority of 5’-ends to a unique position on the small genomes of the bacteria in this study. However, longer Illumina reads (and thus longer mapping sequences) can be used if the TSSs are in repeated regions or if large-genome organisms, such as humans, are being examined. h The in silico pipeline input consists of stranded RNA-seq reads for one or multiple biological replicates in FASTQ format. Each replicate includes a FASTQ for the -RppH pool and another for the + RppH pool. i The FASTQ files go through EMOTE-conv software [ 51 ] that parses the reads, aligns them to the genome, and perform the quantification. Thus, for each genomic position we obtain the number of reads whose first nucleotide align at this genomic position, and on which strand it maps. The counts are further corrected for PCR biases by looking at the unique molecular identifiers (UMIs) sequences available in the unaligned part of the EMOTE read. j Quantification counts obtained for + RppH and -RppH pools are compared through a beta-binomial model that tests whether the identified 5’ ends in the + RppH pool is significantly enriched over the identified 5’ ends in the -RppH pool at a given position. The process results in a p-value that reflects our confidence in the genomic position to be enriched in the + RppH pool of the biological replicate. k The p-values of all the biological replicates are combined into a single p-value with Fisher’s method. l and m To correct the p-values for multiple testing across all genomic positions, the false discovery rate (FDR) is evaluated and only those with a FDR ≤ 0.01 are considered to be TSSs. Note also that for the FDR is only calculated for genomic positions with at least 5 detected ligation-events in at least one of the + RppH pools (UMI ≥ 5). n The TSSs then enter an annotation process that retrieve their surrounding sequence and downstream ORFs. TSSs separated by less than 5 bp are clustered together. Finally, to draw a global picture of operon structures, an independent detection of transcription terminators is operated with the software TransTermHP [ 39 ]. o Sequence of the RNA oligo Rp6 and a typical Illumina sequencing read from a TSS-EMOTE experiment. The Recognition Sequence serves as priming site for the PCR in panel F. UMI: The randomly incorporated nucleotides in the Rp6 oligo that serves to whether Illumina reads with identical Mapping Sequences originate from separate ligation events. CS: Control Sequence. EB: EMOTE barcode to index the Illumina reads. An asterisk indicates the 5’ nucleotide of the original RNA molecule

    Article Snippet: The “+RppH mix” consisted of 1.5 μl water, 2 ul RNA ligase buffer, 2 μl 10 mM ATP, 1 μl Murine RNase inhibitor, 2 μl RNA ligase 1, and 2 μl RppH (New England Biolabs).

    Techniques: In Silico, Purification, Ligation, Sequencing, Polymerase Chain Reaction, RNA Sequencing Assay, Software