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  • 99
    New England Biolabs t4 rna ligase buffer
    DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using <t>T4</t> RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.
    T4 Rna Ligase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 526 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t4 rna ligase buffer
    5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that <t>T4</t> RNA ligase was omitted. ( C ) The strategy in A ).
    T4 Rna Ligase Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    TaKaRa t4 rna ligase buffer
    Gel electrophoresis pattern of mRNA-linker ligation. The ligation products reacted with or without prRT- DNA oligomer used as a blocker of the 3'-end of mRNA were electrophoresis on 8 M urea 8 % PAGE at 65 °C and were visualized with fluorescence of (A) SYBR Green II and (B) FITC. Lane M: DNA ladder, Lane Y: ligation product, Lane L-: negative control, reaction product without DNA-linker, Lane E-: negative control, reaction product without <t>T4</t> RNA ligase. Mobility of the mRNA-linker and the self-ligation product of mRNA are shown to be equivalent.
    T4 Rna Ligase Buffer, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Promega t4 rna ligase buffer
    Gel electrophoresis pattern of mRNA-linker ligation. The ligation products reacted with or without prRT- DNA oligomer used as a blocker of the 3'-end of mRNA were electrophoresis on 8 M urea 8 % PAGE at 65 °C and were visualized with fluorescence of (A) SYBR Green II and (B) FITC. Lane M: DNA ladder, Lane Y: ligation product, Lane L-: negative control, reaction product without DNA-linker, Lane E-: negative control, reaction product without <t>T4</t> RNA ligase. Mobility of the mRNA-linker and the self-ligation product of mRNA are shown to be equivalent.
    T4 Rna Ligase Buffer, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Roche t4 rna ligase buffer
    Analysis of wild-type and mutant virion RNA content by RNA end-labeling. RNA extracted from virions was end-labeled with <t>T4</t> RNA ligase and [ 32 P]pCp, and was analyzed on a denaturing 1% agarose gel. ( A ) RNAs extracted from the same number of wild-type (lane 1) and Ψ − (lane 2) virion particles or from a mock preparation (lane 3). ( B ) RNAs extracted from the same number of PR − (lane 4) and Ψ − PR − (lane 5) virion particles or from a mock preparation (lane 6). Lanes 1, 2, and 3 of B represent 2, 1, and 0.5 pmol of yeast tRNA, respectively, which were labeled by the same technique as the other RNA samples.
    T4 Rna Ligase Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Enzymatics t4 rna ligase 2tr buffer
    Analysis of wild-type and mutant virion RNA content by RNA end-labeling. RNA extracted from virions was end-labeled with <t>T4</t> RNA ligase and [ 32 P]pCp, and was analyzed on a denaturing 1% agarose gel. ( A ) RNAs extracted from the same number of wild-type (lane 1) and Ψ − (lane 2) virion particles or from a mock preparation (lane 3). ( B ) RNAs extracted from the same number of PR − (lane 4) and Ψ − PR − (lane 5) virion particles or from a mock preparation (lane 6). Lanes 1, 2, and 3 of B represent 2, 1, and 0.5 pmol of yeast tRNA, respectively, which were labeled by the same technique as the other RNA samples.
    T4 Rna Ligase 2tr Buffer, supplied by Enzymatics, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t4 rna ligase 1 buffer
    Analysis of wild-type and mutant virion RNA content by RNA end-labeling. RNA extracted from virions was end-labeled with <t>T4</t> RNA ligase and [ 32 P]pCp, and was analyzed on a denaturing 1% agarose gel. ( A ) RNAs extracted from the same number of wild-type (lane 1) and Ψ − (lane 2) virion particles or from a mock preparation (lane 3). ( B ) RNAs extracted from the same number of PR − (lane 4) and Ψ − PR − (lane 5) virion particles or from a mock preparation (lane 6). Lanes 1, 2, and 3 of B represent 2, 1, and 0.5 pmol of yeast tRNA, respectively, which were labeled by the same technique as the other RNA samples.
    T4 Rna Ligase 1 Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    t4 rna ligase 1 buffer - by Bioz Stars, 2020-08
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    99
    New England Biolabs t4 rna ligase 2 truncated buffer
    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 Truncated Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using T4 RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.

    Journal: Genome Research

    Article Title: Strand-specific deep sequencing of the transcriptome

    doi: 10.1101/gr.094318.109

    Figure Lengend Snippet: DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using T4 RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.

    Article Snippet: We incubated the following reaction mixture for 30 min at 37°C: 10 μL of sample, 1 μL of 10× T4 RNA ligase buffer (as fresh ATP supply), 10 U of polynucleotide kinase (New England BioLabs), 3 μL of RNase free water.

    Techniques: De-Phosphorylation Assay, Ligation, Polyacrylamide Gel Electrophoresis, Selection, Amplification, Polymerase Chain Reaction, Sequencing

    tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with

    Journal:

    Article Title: Loss of a Universal tRNA Feature ▿

    doi: 10.1128/JB.01203-06

    Figure Lengend Snippet: tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with

    Article Snippet: For rephosphorylation, dephosphorylated RNA was incubated at 125 ng/μl for 50 min at 37°C in RNA ligase buffer with 0.5 U of T4 polynucleotide kinase (New England Biolabs)/μl, purifying the product RNA as described above.

    Techniques: Sequencing, Ligation, Reverse Transcription Polymerase Chain Reaction

    Library preparation using the CapSMART method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Phosphorylation to add mono-phosphate to the non-capped 5′ end molecules using T4 Polynucleotide Kinase. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides ( Table 2 : STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.

    Journal: PLoS ONE

    Article Title: Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform

    doi: 10.1371/journal.pone.0101812

    Figure Lengend Snippet: Library preparation using the CapSMART method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Phosphorylation to add mono-phosphate to the non-capped 5′ end molecules using T4 Polynucleotide Kinase. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides ( Table 2 : STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.

    Article Snippet: The products were then treated with T4 Polynucleotide Kinase to add mono-phosphate to non-capped mRNA to ready it for ligation; a reaction mixture consisting of 1 µl of T4 Polynucleotide Kinase (Fermentas, # EK0032), 2 µl of RNA Ligase Reaction Buffer (New England Biolabs), 0.5 µl of RNaseOUT (Invitrogen, #10777-019), 1 µl of 100 mM ATP solution (Fermentas, #R0441), and 15.5 µl of alkaline phosphatase-treated RNA was incubated for 30 minutes at 37°C.

    Techniques: De-Phosphorylation Assay, Ligation, Amplification, Polymerase Chain Reaction, Sequencing

    5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).

    Journal: RNA

    Article Title: Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)

    doi: 10.1261/rna.5247704

    Figure Lengend Snippet: 5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).

    Article Snippet: The solution was brought to 100 μL total volume containing 1× T4 RNA ligase buffer (see above), 50 μM ATP, and 40–200 units of T4 RNA ligase (MBI Fermentas).

    Techniques: Electrophoretic Mobility Shift Assay, Blocking Assay, Polyacrylamide Gel Electrophoresis, Radioactivity, Positive Control

    5′-Adenylated RNA. ( A ) The structure of 5′-AppRNA. X is the 5′-terminal nucleotide of the RNA substrate before adenylation. ( B ) The T4 RNA ligase mechanism, showing the 5′-AppRNA intermediate 2 . X and X′ may be any nucleotides. ( C ) Nucleophilic displacement reaction on 5′-triphosphorylated RNA (5′-pppRNA). Nu, nucleophile. The 5′-terminal nucleotide of the RNA is shown as guanosine G because 5′-triphosphorylated RNAs are most typically prepared by in vitro transcription, which introduces G at this position. The nucleophilic substitution reaction on 5′-AppRNA is analogous, except with displacement of AMP instead of PP i (cf. 2 → 3 in B ).

    Journal: RNA

    Article Title: Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)

    doi: 10.1261/rna.5247704

    Figure Lengend Snippet: 5′-Adenylated RNA. ( A ) The structure of 5′-AppRNA. X is the 5′-terminal nucleotide of the RNA substrate before adenylation. ( B ) The T4 RNA ligase mechanism, showing the 5′-AppRNA intermediate 2 . X and X′ may be any nucleotides. ( C ) Nucleophilic displacement reaction on 5′-triphosphorylated RNA (5′-pppRNA). Nu, nucleophile. The 5′-terminal nucleotide of the RNA is shown as guanosine G because 5′-triphosphorylated RNAs are most typically prepared by in vitro transcription, which introduces G at this position. The nucleophilic substitution reaction on 5′-AppRNA is analogous, except with displacement of AMP instead of PP i (cf. 2 → 3 in B ).

    Article Snippet: The solution was brought to 100 μL total volume containing 1× T4 RNA ligase buffer (see above), 50 μM ATP, and 40–200 units of T4 RNA ligase (MBI Fermentas).

    Techniques: In Vitro

    Possible reaction products from 5′-adenylation of an RNA substrate with T4 RNA ligase and ATP. 5′-monophosphate and 5′-adenyl pyrophosphate termini are abbreviated p and App, respectively. The 5′-to-3′ polarity of each strand is shown by an arrowhead pointing in the 3′-direction. The desired 5′-AppRNA is boxed, and the three possible side reactions starting from 5′-AppRNA are illustrated (circularization, oligomerization, and blocking oligo ligation). The abbreviations used for the other products in the remaining figures of this article are given in boldface within parentheses. For the oligomerization reaction, the RNA substrate that does not provide the reactive 5′-App may itself have either 5′-p or 5′-App. Therefore, two different oligomerization products of any given nucleotide length are possible; only one is shown here.

    Journal: RNA

    Article Title: Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)

    doi: 10.1261/rna.5247704

    Figure Lengend Snippet: Possible reaction products from 5′-adenylation of an RNA substrate with T4 RNA ligase and ATP. 5′-monophosphate and 5′-adenyl pyrophosphate termini are abbreviated p and App, respectively. The 5′-to-3′ polarity of each strand is shown by an arrowhead pointing in the 3′-direction. The desired 5′-AppRNA is boxed, and the three possible side reactions starting from 5′-AppRNA are illustrated (circularization, oligomerization, and blocking oligo ligation). The abbreviations used for the other products in the remaining figures of this article are given in boldface within parentheses. For the oligomerization reaction, the RNA substrate that does not provide the reactive 5′-App may itself have either 5′-p or 5′-App. Therefore, two different oligomerization products of any given nucleotide length are possible; only one is shown here.

    Article Snippet: The solution was brought to 100 μL total volume containing 1× T4 RNA ligase buffer (see above), 50 μM ATP, and 40–200 units of T4 RNA ligase (MBI Fermentas).

    Techniques: Blocking Assay, Ligation

    Gel electrophoresis pattern of mRNA-linker ligation. The ligation products reacted with or without prRT- DNA oligomer used as a blocker of the 3'-end of mRNA were electrophoresis on 8 M urea 8 % PAGE at 65 °C and were visualized with fluorescence of (A) SYBR Green II and (B) FITC. Lane M: DNA ladder, Lane Y: ligation product, Lane L-: negative control, reaction product without DNA-linker, Lane E-: negative control, reaction product without T4 RNA ligase. Mobility of the mRNA-linker and the self-ligation product of mRNA are shown to be equivalent.

    Journal: International Journal of Biological Sciences

    Article Title: An mRNA-protein Fusion at N-terminus for Evolutionary Protein Engineering

    doi:

    Figure Lengend Snippet: Gel electrophoresis pattern of mRNA-linker ligation. The ligation products reacted with or without prRT- DNA oligomer used as a blocker of the 3'-end of mRNA were electrophoresis on 8 M urea 8 % PAGE at 65 °C and were visualized with fluorescence of (A) SYBR Green II and (B) FITC. Lane M: DNA ladder, Lane Y: ligation product, Lane L-: negative control, reaction product without DNA-linker, Lane E-: negative control, reaction product without T4 RNA ligase. Mobility of the mRNA-linker and the self-ligation product of mRNA are shown to be equivalent.

    Article Snippet: Ligation between the mRNA and the hydrazide-linker The mRNA (2 µM) was hybridized to the DNA moiety of the hydrazide-linker (4 µM) and prRT- (4 µM) by heating at 95 °C and cooling to 25 °C in 50 µl of T4 RNA ligase buffer (Takara Bio) and ligation reaction was started by adding T4 RNA ligase (40 U, Takara Bio) and ribonuclease inhibitor (40 U, Takara Bio).

    Techniques: Nucleic Acid Electrophoresis, Ligation, Electrophoresis, Polyacrylamide Gel Electrophoresis, Fluorescence, SYBR Green Assay, Negative Control

    Screening cycle of the mRNA-protein fusion in this study. The dsDNA library is transcribed to mRNA. The mRNA is hybridized to the DNA moiety of the linker having hydrazide group and ligated with T4 RNA ligase. Hydrazide group of the ligated product and acetyl group of the phenylalanine derivative that is acylated to sup tRNA are ligated chemically and the modified mRNA is translated. The modified aminoacyl sup tRNA tends to occupy the A-site of ribosome at UAG codon inserted near downstream of initiation codon and the phenylalanine is incorporated into the growing peptide. Thus, linkage between N-terminus of the nascent peptide and 5'-terminus of its mRNA is achieved. Screening of mRNA-peptide fusion library according to property of the displayed peptide and amplify the genotype molecules of the screened fusions by RT-PCR.

    Journal: International Journal of Biological Sciences

    Article Title: An mRNA-protein Fusion at N-terminus for Evolutionary Protein Engineering

    doi:

    Figure Lengend Snippet: Screening cycle of the mRNA-protein fusion in this study. The dsDNA library is transcribed to mRNA. The mRNA is hybridized to the DNA moiety of the linker having hydrazide group and ligated with T4 RNA ligase. Hydrazide group of the ligated product and acetyl group of the phenylalanine derivative that is acylated to sup tRNA are ligated chemically and the modified mRNA is translated. The modified aminoacyl sup tRNA tends to occupy the A-site of ribosome at UAG codon inserted near downstream of initiation codon and the phenylalanine is incorporated into the growing peptide. Thus, linkage between N-terminus of the nascent peptide and 5'-terminus of its mRNA is achieved. Screening of mRNA-peptide fusion library according to property of the displayed peptide and amplify the genotype molecules of the screened fusions by RT-PCR.

    Article Snippet: Ligation between the mRNA and the hydrazide-linker The mRNA (2 µM) was hybridized to the DNA moiety of the hydrazide-linker (4 µM) and prRT- (4 µM) by heating at 95 °C and cooling to 25 °C in 50 µl of T4 RNA ligase buffer (Takara Bio) and ligation reaction was started by adding T4 RNA ligase (40 U, Takara Bio) and ribonuclease inhibitor (40 U, Takara Bio).

    Techniques: Modification, Reverse Transcription Polymerase Chain Reaction

    Analysis of wild-type and mutant virion RNA content by RNA end-labeling. RNA extracted from virions was end-labeled with T4 RNA ligase and [ 32 P]pCp, and was analyzed on a denaturing 1% agarose gel. ( A ) RNAs extracted from the same number of wild-type (lane 1) and Ψ − (lane 2) virion particles or from a mock preparation (lane 3). ( B ) RNAs extracted from the same number of PR − (lane 4) and Ψ − PR − (lane 5) virion particles or from a mock preparation (lane 6). Lanes 1, 2, and 3 of B represent 2, 1, and 0.5 pmol of yeast tRNA, respectively, which were labeled by the same technique as the other RNA samples.

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

    Article Title: RNA is a structural element in retrovirus particles

    doi: 10.1073/pnas.091000398

    Figure Lengend Snippet: Analysis of wild-type and mutant virion RNA content by RNA end-labeling. RNA extracted from virions was end-labeled with T4 RNA ligase and [ 32 P]pCp, and was analyzed on a denaturing 1% agarose gel. ( A ) RNAs extracted from the same number of wild-type (lane 1) and Ψ − (lane 2) virion particles or from a mock preparation (lane 3). ( B ) RNAs extracted from the same number of PR − (lane 4) and Ψ − PR − (lane 5) virion particles or from a mock preparation (lane 6). Lanes 1, 2, and 3 of B represent 2, 1, and 0.5 pmol of yeast tRNA, respectively, which were labeled by the same technique as the other RNA samples.

    Article Snippet: RNA extracted from viral particles or BHK cells was dissolved in 10 μl of RNase-free water and mixed with 17 μl of a buffer containing 10% DMSO (Sigma), 1× T4 RNA ligase buffer (Roche), 4 units of recombinant RNasin (Promega), and 10 μg/ml BSA.

    Techniques: Mutagenesis, End Labeling, Labeling, Agarose Gel Electrophoresis

    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