t4 rna ligase buffer  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97

    Structured Review

    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: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase buffer/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase buffer - by Bioz Stars, 2022-05
    97/100 stars

    Images

    1) Product Images from "Strand-specific deep sequencing of the transcriptome"

    Article Title: Strand-specific deep sequencing of the transcriptome

    Journal: Genome Research

    doi: 10.1101/gr.094318.109

    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.
    Figure Legend 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.

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

    2) Product Images from "Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0101812

    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.
    Figure Legend 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.

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

    3) 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

    4) Product Images from "Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts"

    Article Title: Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts

    Journal: eLife

    doi: 10.7554/eLife.27024

    In vitro optimization of RNA-to-DNA ligation conditions. Upper panel , Ten pmols of 17-nt adenylated ssDNA oligonucleotide (Universal App DNA, CTGTAGGCACCATCAAT) was incubated with 5 pmols of a 17nt ssRNA test probe (TTTCGTTGGAAGCGGGA) in 1x NEB T4 RNA Ligase Buffer with the indicated ligase (NEB Thermostable 5’ AppDNA/RNA ligase (Therm 5' Ligase), NEB T4 Rnl2tr K227Q Ligase (trT4K) or NEB T4 Rnl2tr R55K, K227Q ligase (trT4KQ)) and/or supplements (PEG, BSA, ATP, RNaseOUT). Products were then analyzed using denaturing polyacrylamide gel electrophoresis using a combination of NEB microRNA and low range ssRNA ladders and stained with SYBR-gold. Bands were quantified and the percent product was calculated using (shifted / (total * 0.66)) to account for the molar excess of DNA over RNA. No adjustment was made to account for preferential staining of ssDNA over ssRNA. Residual signal is expected in the lower band owing to the molar excess of DNA over RNA. A high molecular weight band is visible in the Therm 5’ Ligase lane, which most likely consists of high molecular weight concatemers of the AppDNA substrate caused by incomplete 3’ blocking of these oligos or removal of the 3’ block by the Therm 5’ Ligase. This experiment was performed once.
    Figure Legend Snippet: In vitro optimization of RNA-to-DNA ligation conditions. Upper panel , Ten pmols of 17-nt adenylated ssDNA oligonucleotide (Universal App DNA, CTGTAGGCACCATCAAT) was incubated with 5 pmols of a 17nt ssRNA test probe (TTTCGTTGGAAGCGGGA) in 1x NEB T4 RNA Ligase Buffer with the indicated ligase (NEB Thermostable 5’ AppDNA/RNA ligase (Therm 5' Ligase), NEB T4 Rnl2tr K227Q Ligase (trT4K) or NEB T4 Rnl2tr R55K, K227Q ligase (trT4KQ)) and/or supplements (PEG, BSA, ATP, RNaseOUT). Products were then analyzed using denaturing polyacrylamide gel electrophoresis using a combination of NEB microRNA and low range ssRNA ladders and stained with SYBR-gold. Bands were quantified and the percent product was calculated using (shifted / (total * 0.66)) to account for the molar excess of DNA over RNA. No adjustment was made to account for preferential staining of ssDNA over ssRNA. Residual signal is expected in the lower band owing to the molar excess of DNA over RNA. A high molecular weight band is visible in the Therm 5’ Ligase lane, which most likely consists of high molecular weight concatemers of the AppDNA substrate caused by incomplete 3’ blocking of these oligos or removal of the 3’ block by the Therm 5’ Ligase. This experiment was performed once.

    Techniques Used: In Vitro, DNA Ligation, Incubation, Polyacrylamide Gel Electrophoresis, Staining, Molecular Weight, Blocking Assay

    5) 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

    Schematic workflow of the LOTTE-seq procedure. (A) A DNA hairpin-oligonucleotide (green) with a 3ʹ-TGGN overhang hybridizes to the tRNA 3ʹ-CCA end (tRNA in blue). T4 DNA ligase fuses the 3ʹ-end of the CCA terminus to the phosphorylated 5ʹ end of the adapter. (B) The tRNA is reverse transcribed with parts of the hairpin oligonucleotide serving as primer binding site. Secondary structure and modified bases can lead to premature RT stops and partial cDNA (yellow). (C) Using T4 RNA ligase I, a 5ʹ-phosphorylated and 3ʹ-blocked second adapter (red) is fused to the 3ʹ-end of the cDNA, leading to the generation of cDNA product with adapters on both sides (red and green). (D) This product is PCR-amplified with indexed primers binding to the adapter overhang sequences. (E) The cDNA library consisting of full-length as well as prematurely terminated tRNA sequences is analysed by high-throughput sequencing.
    Figure Legend Snippet: Schematic workflow of the LOTTE-seq procedure. (A) A DNA hairpin-oligonucleotide (green) with a 3ʹ-TGGN overhang hybridizes to the tRNA 3ʹ-CCA end (tRNA in blue). T4 DNA ligase fuses the 3ʹ-end of the CCA terminus to the phosphorylated 5ʹ end of the adapter. (B) The tRNA is reverse transcribed with parts of the hairpin oligonucleotide serving as primer binding site. Secondary structure and modified bases can lead to premature RT stops and partial cDNA (yellow). (C) Using T4 RNA ligase I, a 5ʹ-phosphorylated and 3ʹ-blocked second adapter (red) is fused to the 3ʹ-end of the cDNA, leading to the generation of cDNA product with adapters on both sides (red and green). (D) This product is PCR-amplified with indexed primers binding to the adapter overhang sequences. (E) The cDNA library consisting of full-length as well as prematurely terminated tRNA sequences is analysed by high-throughput sequencing.

    Techniques Used: Binding Assay, Modification, Polymerase Chain Reaction, Amplification, cDNA Library Assay, Next-Generation Sequencing

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1049

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

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

    7) 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

    8) Product Images from "Chromatin-associated RNA sequencing (ChAR-seq)"

    Article Title: Chromatin-associated RNA sequencing (ChAR-seq)

    Journal: Current protocols in molecular biology

    doi: 10.1002/cpmb.87

    : briefly, (1) 5 adenylation (5’-App) that allows specific ligation of ssDNA to ssRNA with a mutated T4 RNA ligase; (2) random barcode (blue); (3) DpnII recognition site (pink); (4) biotin for isolation (purple); (5) PacI recognition site (orange). Gray lines represent restriction enzyme cleavage patterns. (B) Schematic of the final dsDNA molecule that results from the ChAR-seq procedure. RNA and DNA in close proximity will ligate to the bridge linker molecule as shown. Red represents cDNA derived from the ssRNA and green represents genomic DNA. Dashed lines on either side represent extended sequence of varying length.
    Figure Legend Snippet: : briefly, (1) 5 adenylation (5’-App) that allows specific ligation of ssDNA to ssRNA with a mutated T4 RNA ligase; (2) random barcode (blue); (3) DpnII recognition site (pink); (4) biotin for isolation (purple); (5) PacI recognition site (orange). Gray lines represent restriction enzyme cleavage patterns. (B) Schematic of the final dsDNA molecule that results from the ChAR-seq procedure. RNA and DNA in close proximity will ligate to the bridge linker molecule as shown. Red represents cDNA derived from the ssRNA and green represents genomic DNA. Dashed lines on either side represent extended sequence of varying length.

    Techniques Used: Ligation, Isolation, Derivative Assay, Sequencing

    9) Product Images from "A conserved long intergenic non-coding RNA containing snoRNA sequences, lncCOBRA1, affects Arabidopsis germination and development"

    Article Title: A conserved long intergenic non-coding RNA containing snoRNA sequences, lncCOBRA1, affects Arabidopsis germination and development

    Journal: bioRxiv

    doi: 10.1101/2021.11.28.470209

    Post-transcriptional processing of lncCOBRA1 (A) Diagram of lncCOBRA1 ( AT1G05913 ) locus. Gray arrows represent the two snoRNAs annotated within lncCOBRA1. Red arrows represent the two primers used for 5’ RACE and red triangle represents the 5’ end identified by 5’ RACE PCR in Figure 2B . Blue arrow represents the primer used for 3’ RACE. Blue triangles represent the 3’ most end identified through Sanger sequencing 14 colonies. (B) Three biological replicates of 5’ RACE with primers indicated in Figure 2B . Red triangles represent the two major bands of PCR product. Ladder is 1 kb+. (C) PCR results from 3’ RACE in Col-0 5-day-old seedlings. -/+ T4 RNA ligase, -/+ SuperScript II. Ladder is 1 kb+.
    Figure Legend Snippet: Post-transcriptional processing of lncCOBRA1 (A) Diagram of lncCOBRA1 ( AT1G05913 ) locus. Gray arrows represent the two snoRNAs annotated within lncCOBRA1. Red arrows represent the two primers used for 5’ RACE and red triangle represents the 5’ end identified by 5’ RACE PCR in Figure 2B . Blue arrow represents the primer used for 3’ RACE. Blue triangles represent the 3’ most end identified through Sanger sequencing 14 colonies. (B) Three biological replicates of 5’ RACE with primers indicated in Figure 2B . Red triangles represent the two major bands of PCR product. Ladder is 1 kb+. (C) PCR results from 3’ RACE in Col-0 5-day-old seedlings. -/+ T4 RNA ligase, -/+ SuperScript II. Ladder is 1 kb+.

    Techniques Used: Polymerase Chain Reaction, Sequencing

    10) Product Images from "TAC-seq: targeted DNA and RNA sequencing for precise biomarker molecule counting"

    Article Title: TAC-seq: targeted DNA and RNA sequencing for precise biomarker molecule counting

    Journal: bioRxiv

    doi: 10.1101/295253

    Principle and technical parameters of TAC-seq. ( A ) Schematic diagram of the assay to detect specific mRNA or cell-free DNA. Target-specific DNA oligonucleotide detector probes hybridize under stringent conditions to the studied cDNA or cfDNA. Both detector oligonucleotides consist of a specific 27-bp region (green), 4-bp unique molecular identifier (UMI) motif (NNNN) and universal sequences (purple and orange). The right detector oligonucleotide is 5’ phosphorylated. After rigorous hybridization, the pair of detector probes is ligated using a thermostable ligase under stringent conditions. Next, the ligated detectors complexed with the target region are captured with magnetic beads and PCR amplified to introduce sample-specific barcodes and other common motifs that are required for single-read NGS. ( B ) Spearman correlation analysis of the input and detected ERCC synthetic spike-in mRNA molecules at UMI threshold 4 (UMI=4). UMI threshold is defined as the number of detected unique UMI sequences. For example, UMI=4 indicates that a certain UMI motif is detected at least four times. UMIs are valuable only if the number of UMI combinations (8-bp UMI provides 65,536 variants, for example) is substantially larger than the sum of the target molecules in the studied sample. ( C ) Bar plot of Spearman’s correlation analysis of the ERCC input and detected molecules at different UMI thresholds. ( D ) Reproducibility of seven technical ERCC replicates (seven different icons on plot) of 22 spike-in molecules at UMI=4.
    Figure Legend Snippet: Principle and technical parameters of TAC-seq. ( A ) Schematic diagram of the assay to detect specific mRNA or cell-free DNA. Target-specific DNA oligonucleotide detector probes hybridize under stringent conditions to the studied cDNA or cfDNA. Both detector oligonucleotides consist of a specific 27-bp region (green), 4-bp unique molecular identifier (UMI) motif (NNNN) and universal sequences (purple and orange). The right detector oligonucleotide is 5’ phosphorylated. After rigorous hybridization, the pair of detector probes is ligated using a thermostable ligase under stringent conditions. Next, the ligated detectors complexed with the target region are captured with magnetic beads and PCR amplified to introduce sample-specific barcodes and other common motifs that are required for single-read NGS. ( B ) Spearman correlation analysis of the input and detected ERCC synthetic spike-in mRNA molecules at UMI threshold 4 (UMI=4). UMI threshold is defined as the number of detected unique UMI sequences. For example, UMI=4 indicates that a certain UMI motif is detected at least four times. UMIs are valuable only if the number of UMI combinations (8-bp UMI provides 65,536 variants, for example) is substantially larger than the sum of the target molecules in the studied sample. ( C ) Bar plot of Spearman’s correlation analysis of the ERCC input and detected molecules at different UMI thresholds. ( D ) Reproducibility of seven technical ERCC replicates (seven different icons on plot) of 22 spike-in molecules at UMI=4.

    Techniques Used: Hybridization, Magnetic Beads, Polymerase Chain Reaction, Amplification, Introduce, Next-Generation Sequencing

    Comparison of the overall predictions for mRNA TAC-seq assay. ( A ) Principal component analysis of the full transcriptome RNA-seq, high-coverage TAC-seq and low-coverage TAC-seq of 10 endometrial samples. The first principal component (PC1) describes most of the sample variability and correlates most with the receptivity status. Blue dots represent pre-receptive and red dots receptive human endometrial samples. One separate pre-receptive sample (indicated with an asterisk) represents the same sample that clusters differently in the heatmap analysis (below) and is therefore a potential biological outlier. ( B ) Heatmaps of the full transcriptome RNA-seq, high-coverage- and low-coverage TAC-seq show the sensitivity to distinguish different endometrial samples according to their receptivity. One pre-receptive sample (indicated with an asterisk) shares the expression profile and clusters together with receptive samples in all three comparisons. Pre-receptive samples are labelled blue and receptive red. Detailed heatmaps are presented in Supplemental Fig. S3 together with housekeeping genes that demonstrate a lack of fluctuation of the pre-receptive and receptive biopsies. High-coverage TAC-seq data are presented at UMI=2 and low-coverage data at UMI=1 on PCA and heatmaps. The data are plotted as row-wise scaled log-transformed counts per million (CPM) values. The samples are hierarchically clustered column-wise using Pearson correlation. The genes are ordered row-wise according to the RNA-seq clustering results using Euclidean distance. Fewer genes are found expressed with a low-coverage compared to RNA-seq and high-coverage TAC-seq.
    Figure Legend Snippet: Comparison of the overall predictions for mRNA TAC-seq assay. ( A ) Principal component analysis of the full transcriptome RNA-seq, high-coverage TAC-seq and low-coverage TAC-seq of 10 endometrial samples. The first principal component (PC1) describes most of the sample variability and correlates most with the receptivity status. Blue dots represent pre-receptive and red dots receptive human endometrial samples. One separate pre-receptive sample (indicated with an asterisk) represents the same sample that clusters differently in the heatmap analysis (below) and is therefore a potential biological outlier. ( B ) Heatmaps of the full transcriptome RNA-seq, high-coverage- and low-coverage TAC-seq show the sensitivity to distinguish different endometrial samples according to their receptivity. One pre-receptive sample (indicated with an asterisk) shares the expression profile and clusters together with receptive samples in all three comparisons. Pre-receptive samples are labelled blue and receptive red. Detailed heatmaps are presented in Supplemental Fig. S3 together with housekeeping genes that demonstrate a lack of fluctuation of the pre-receptive and receptive biopsies. High-coverage TAC-seq data are presented at UMI=2 and low-coverage data at UMI=1 on PCA and heatmaps. The data are plotted as row-wise scaled log-transformed counts per million (CPM) values. The samples are hierarchically clustered column-wise using Pearson correlation. The genes are ordered row-wise according to the RNA-seq clustering results using Euclidean distance. Fewer genes are found expressed with a low-coverage compared to RNA-seq and high-coverage TAC-seq.

    Techniques Used: RNA Sequencing Assay, Expressing, Transformation Assay

    11) 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

    Schematic workflow of the LOTTE-seq procedure. (A) A DNA hairpin-oligonucleotide (green) with a 3ʹ-TGGN overhang hybridizes to the tRNA 3ʹ-CCA end (tRNA in blue). T4 DNA ligase fuses the 3ʹ-end of the CCA terminus to the phosphorylated 5ʹ end of the adapter. (B) The tRNA is reverse transcribed with parts of the hairpin oligonucleotide serving as primer binding site. Secondary structure and modified bases can lead to premature RT stops and partial cDNA (yellow). (C) Using T4 RNA ligase I, a 5ʹ-phosphorylated and 3ʹ-blocked second adapter (red) is fused to the 3ʹ-end of the cDNA, leading to the generation of cDNA product with adapters on both sides (red and green). (D) This product is PCR-amplified with indexed primers binding to the adapter overhang sequences. (E) The cDNA library consisting of full-length as well as prematurely terminated tRNA sequences is analysed by high-throughput sequencing.
    Figure Legend Snippet: Schematic workflow of the LOTTE-seq procedure. (A) A DNA hairpin-oligonucleotide (green) with a 3ʹ-TGGN overhang hybridizes to the tRNA 3ʹ-CCA end (tRNA in blue). T4 DNA ligase fuses the 3ʹ-end of the CCA terminus to the phosphorylated 5ʹ end of the adapter. (B) The tRNA is reverse transcribed with parts of the hairpin oligonucleotide serving as primer binding site. Secondary structure and modified bases can lead to premature RT stops and partial cDNA (yellow). (C) Using T4 RNA ligase I, a 5ʹ-phosphorylated and 3ʹ-blocked second adapter (red) is fused to the 3ʹ-end of the cDNA, leading to the generation of cDNA product with adapters on both sides (red and green). (D) This product is PCR-amplified with indexed primers binding to the adapter overhang sequences. (E) The cDNA library consisting of full-length as well as prematurely terminated tRNA sequences is analysed by high-throughput sequencing.

    Techniques Used: Binding Assay, Modification, Polymerase Chain Reaction, Amplification, cDNA Library Assay, Next-Generation Sequencing

    12) Product Images from "Single Nucleotide Resolution RNA–Protein Cross-Linking Mass Spectrometry: A Simple Extension of the CLIR-MS Workflow"

    Article Title: Single Nucleotide Resolution RNA–Protein Cross-Linking Mass Spectrometry: A Simple Extension of the CLIR-MS Workflow

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.1c02384

    Single-Phosphate Labeling (A) Desired product with a labeled phosphate between G6 and U7 of the extended FBE sequence. (B) A single heavy-labeled phosphate is introduced into an RNA by labeling the 5′-end of the 3′-segment (blue) with 18 O 4 -γ-ATP and subsequent ligation to the 5′-segment (green) using T4 RNA ligase. The 3′-RNA is blocked at the 3′-end with a C3-spacer to prevent undesired ligation side products. (C) Simulation of isotopic distribution 33 of a cross-linked peptide (PEPTIDER cross-linked to a GU dinucleotide, doubly charged) for a 1:1 mixture of light and heavy ATP using single-phosphate labeling.
    Figure Legend Snippet: Single-Phosphate Labeling (A) Desired product with a labeled phosphate between G6 and U7 of the extended FBE sequence. (B) A single heavy-labeled phosphate is introduced into an RNA by labeling the 5′-end of the 3′-segment (blue) with 18 O 4 -γ-ATP and subsequent ligation to the 5′-segment (green) using T4 RNA ligase. The 3′-RNA is blocked at the 3′-end with a C3-spacer to prevent undesired ligation side products. (C) Simulation of isotopic distribution 33 of a cross-linked peptide (PEPTIDER cross-linked to a GU dinucleotide, doubly charged) for a 1:1 mixture of light and heavy ATP using single-phosphate labeling.

    Techniques Used: Labeling, Sequencing, Ligation

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97
    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: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase buffer/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase buffer - by Bioz Stars, 2022-05
    97/100 stars
      Buy from Supplier

    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

    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

    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

    In vitro optimization of RNA-to-DNA ligation conditions. Upper panel , Ten pmols of 17-nt adenylated ssDNA oligonucleotide (Universal App DNA, CTGTAGGCACCATCAAT) was incubated with 5 pmols of a 17nt ssRNA test probe (TTTCGTTGGAAGCGGGA) in 1x NEB T4 RNA Ligase Buffer with the indicated ligase (NEB Thermostable 5’ AppDNA/RNA ligase (Therm 5' Ligase), NEB T4 Rnl2tr K227Q Ligase (trT4K) or NEB T4 Rnl2tr R55K, K227Q ligase (trT4KQ)) and/or supplements (PEG, BSA, ATP, RNaseOUT). Products were then analyzed using denaturing polyacrylamide gel electrophoresis using a combination of NEB microRNA and low range ssRNA ladders and stained with SYBR-gold. Bands were quantified and the percent product was calculated using (shifted / (total * 0.66)) to account for the molar excess of DNA over RNA. No adjustment was made to account for preferential staining of ssDNA over ssRNA. Residual signal is expected in the lower band owing to the molar excess of DNA over RNA. A high molecular weight band is visible in the Therm 5’ Ligase lane, which most likely consists of high molecular weight concatemers of the AppDNA substrate caused by incomplete 3’ blocking of these oligos or removal of the 3’ block by the Therm 5’ Ligase. This experiment was performed once.

    Journal: eLife

    Article Title: Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts

    doi: 10.7554/eLife.27024

    Figure Lengend Snippet: In vitro optimization of RNA-to-DNA ligation conditions. Upper panel , Ten pmols of 17-nt adenylated ssDNA oligonucleotide (Universal App DNA, CTGTAGGCACCATCAAT) was incubated with 5 pmols of a 17nt ssRNA test probe (TTTCGTTGGAAGCGGGA) in 1x NEB T4 RNA Ligase Buffer with the indicated ligase (NEB Thermostable 5’ AppDNA/RNA ligase (Therm 5' Ligase), NEB T4 Rnl2tr K227Q Ligase (trT4K) or NEB T4 Rnl2tr R55K, K227Q ligase (trT4KQ)) and/or supplements (PEG, BSA, ATP, RNaseOUT). Products were then analyzed using denaturing polyacrylamide gel electrophoresis using a combination of NEB microRNA and low range ssRNA ladders and stained with SYBR-gold. Bands were quantified and the percent product was calculated using (shifted / (total * 0.66)) to account for the molar excess of DNA over RNA. No adjustment was made to account for preferential staining of ssDNA over ssRNA. Residual signal is expected in the lower band owing to the molar excess of DNA over RNA. A high molecular weight band is visible in the Therm 5’ Ligase lane, which most likely consists of high molecular weight concatemers of the AppDNA substrate caused by incomplete 3’ blocking of these oligos or removal of the 3’ block by the Therm 5’ Ligase. This experiment was performed once.

    Article Snippet: Step 4B ( Optional additional step B ): RNase control Pre-mix and resuspend the pellet in the following 160 µL water 20 µL 10x T4 RNA ligase buffer 10 µL 10 mg/mL RNaseA 10 µL RNaseH (NEB) Incubate at 37C for 4 hr Centrifuge at 2.5 k x g for 2 min, discard supernatant Add 1000 µL DEPC-treated PBS, mix gently Centrifuge at 2.5 k x g for 2 min, discard supernatant Immediately proceed to the next step, with pre-mixed reaction buffer already prepared

    Techniques: In Vitro, DNA Ligation, Incubation, Polyacrylamide Gel Electrophoresis, Staining, Molecular Weight, Blocking Assay