t4 rnl2 reaction buffer  (New England Biolabs)


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    Structured Review

    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: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rnl2 reaction buffer/product/New England Biolabs
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    t4 rnl2 reaction buffer - by Bioz Stars, 2020-05
    96/100 stars

    Images

    1) 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

    Related Articles

    Sample Prep:

    Article Title: The unfolded protein response and endoplasmic reticulum protein targeting machineries converge on the stress sensor IRE1
    Article Snippet: .. Preparation of small RNA cDNA libraries for deep sequencing 1 μL of RA3 RNA 3’ adapter (5’-TGGAATTCTCGGGTGCCAAGG-3’) from the TruSeq RNA small Sample Prep Kit (Illumina) was added to each of the purified RNA samples above, and the mixture was placed at 80°C for 2 min, then placed immediately on ice for another 2 min. 1.5 μL of 10× T4 RNA ligase reaction buffer (500 mM Tris pH 7.5, 100 mM MgCl2 , 10 mM DTT; New England Biolabs) and 4.5 μL 50% PEG 8,000 (New England Biolabs), 1 μL RNase inhibitor (Illumina), and 1 μL (200 units) T4 RNA ligase 2, truncated R55K, K227Q (KQ) mutant (New England Biolabs) were added to each sample and the reactions were incubated at 16°C overnight. .. 12–16 hr later, 0.5 μL of 10× T4 RNA ligase reaction buffer, 1.5 μL of 50% PEG 8,000, 2 μL of RNase-free water and 1 μL (200 units) T4 RNA ligase 2 truncated KQ were added to each sample and the reactions were incubated an additional 2 hr at 25°C.

    Ligation:

    Article Title: Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture
    Article Snippet: .. Ligation Protocol Unless otherwise indicated, ligation was performed by mixing 1.25 µL of 2 µM adenylated adapter, 1 µL of T4 RNA Ligase buffer (New England Biolabs, Ipswich, MA), 5 µL of 50% PEG8000, 1 µL of synthetic target, 0.5 µL of total RNA, 1 µL of T4 RNA Ligase 2 truncated K227Q (New England Biolabs, Ipswich, MA) and water into a 20 µL reaction volume. .. In the experiments where different ligases were investigated, T4 RNA Ligase 2 truncated, T4 RNA Ligase 2 truncated R55K K227Q, and Thermostable 5′ App DNA/RNA Ligase were all obtained from New England Biolabs.

    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 †
    Article Snippet: .. Thermo-denaturation of the RNA prior to the 3′5′ RACE Five micrograms of RNA in a total volume of 20 µl containing the T4 RNA ligase buffer (New England Biolabs, n° B0204S) were incubated for 4 min at 75°C, and then rapidly frozen in a solid CO2 (dry ice)-ethanol bath for 1 min. To allow the samples to thaw slowly, they were then placed on ice for about 15 min, as described in ref. 22, and then 2 µl of T4 RNA ligase was added to start the 3′5′ RNA ligation. ..

    Article Title: Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform
    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. .. Next, 20 µl of T4 Polynucleotide Kinase-treated RNA were incubated with 2.5 µl of nuclease-free water, 1 µl of RNA Ligase Reaction Buffer (New England Biolabs), 4.5 µl of PEG8000 (New England Biolabs), 1 µl of STOP oligo { , STOP1 (50 µM): iGiCiG, STOP2 (50 µM): iCiGiC, STOP Mix (50 µM): mixture of STOP1 and STOP2, synthesized by Metabion, Germany}, and 1 µl of T4 RNA Ligase (New England Biolabs, M0204S) for 16 hours at 16°C to ligate STOP oligos to the non-capped mRNA.

    Mutagenesis:

    Article Title: The unfolded protein response and endoplasmic reticulum protein targeting machineries converge on the stress sensor IRE1
    Article Snippet: .. Preparation of small RNA cDNA libraries for deep sequencing 1 μL of RA3 RNA 3’ adapter (5’-TGGAATTCTCGGGTGCCAAGG-3’) from the TruSeq RNA small Sample Prep Kit (Illumina) was added to each of the purified RNA samples above, and the mixture was placed at 80°C for 2 min, then placed immediately on ice for another 2 min. 1.5 μL of 10× T4 RNA ligase reaction buffer (500 mM Tris pH 7.5, 100 mM MgCl2 , 10 mM DTT; New England Biolabs) and 4.5 μL 50% PEG 8,000 (New England Biolabs), 1 μL RNase inhibitor (Illumina), and 1 μL (200 units) T4 RNA ligase 2, truncated R55K, K227Q (KQ) mutant (New England Biolabs) were added to each sample and the reactions were incubated at 16°C overnight. .. 12–16 hr later, 0.5 μL of 10× T4 RNA ligase reaction buffer, 1.5 μL of 50% PEG 8,000, 2 μL of RNase-free water and 1 μL (200 units) T4 RNA ligase 2 truncated KQ were added to each sample and the reactions were incubated an additional 2 hr at 25°C.

    Purification:

    Article Title: The unfolded protein response and endoplasmic reticulum protein targeting machineries converge on the stress sensor IRE1
    Article Snippet: .. Preparation of small RNA cDNA libraries for deep sequencing 1 μL of RA3 RNA 3’ adapter (5’-TGGAATTCTCGGGTGCCAAGG-3’) from the TruSeq RNA small Sample Prep Kit (Illumina) was added to each of the purified RNA samples above, and the mixture was placed at 80°C for 2 min, then placed immediately on ice for another 2 min. 1.5 μL of 10× T4 RNA ligase reaction buffer (500 mM Tris pH 7.5, 100 mM MgCl2 , 10 mM DTT; New England Biolabs) and 4.5 μL 50% PEG 8,000 (New England Biolabs), 1 μL RNase inhibitor (Illumina), and 1 μL (200 units) T4 RNA ligase 2, truncated R55K, K227Q (KQ) mutant (New England Biolabs) were added to each sample and the reactions were incubated at 16°C overnight. .. 12–16 hr later, 0.5 μL of 10× T4 RNA ligase reaction buffer, 1.5 μL of 50% PEG 8,000, 2 μL of RNase-free water and 1 μL (200 units) T4 RNA ligase 2 truncated KQ were added to each sample and the reactions were incubated an additional 2 hr at 25°C.

    Incubation:

    Article Title: The unfolded protein response and endoplasmic reticulum protein targeting machineries converge on the stress sensor IRE1
    Article Snippet: .. Preparation of small RNA cDNA libraries for deep sequencing 1 μL of RA3 RNA 3’ adapter (5’-TGGAATTCTCGGGTGCCAAGG-3’) from the TruSeq RNA small Sample Prep Kit (Illumina) was added to each of the purified RNA samples above, and the mixture was placed at 80°C for 2 min, then placed immediately on ice for another 2 min. 1.5 μL of 10× T4 RNA ligase reaction buffer (500 mM Tris pH 7.5, 100 mM MgCl2 , 10 mM DTT; New England Biolabs) and 4.5 μL 50% PEG 8,000 (New England Biolabs), 1 μL RNase inhibitor (Illumina), and 1 μL (200 units) T4 RNA ligase 2, truncated R55K, K227Q (KQ) mutant (New England Biolabs) were added to each sample and the reactions were incubated at 16°C overnight. .. 12–16 hr later, 0.5 μL of 10× T4 RNA ligase reaction buffer, 1.5 μL of 50% PEG 8,000, 2 μL of RNase-free water and 1 μL (200 units) T4 RNA ligase 2 truncated KQ were added to each sample and the reactions were incubated an additional 2 hr at 25°C.

    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 †
    Article Snippet: .. Thermo-denaturation of the RNA prior to the 3′5′ RACE Five micrograms of RNA in a total volume of 20 µl containing the T4 RNA ligase buffer (New England Biolabs, n° B0204S) were incubated for 4 min at 75°C, and then rapidly frozen in a solid CO2 (dry ice)-ethanol bath for 1 min. To allow the samples to thaw slowly, they were then placed on ice for about 15 min, as described in ref. 22, and then 2 µl of T4 RNA ligase was added to start the 3′5′ RNA ligation. ..

    Article Title: Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform
    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. .. Next, 20 µl of T4 Polynucleotide Kinase-treated RNA were incubated with 2.5 µl of nuclease-free water, 1 µl of RNA Ligase Reaction Buffer (New England Biolabs), 4.5 µl of PEG8000 (New England Biolabs), 1 µl of STOP oligo { , STOP1 (50 µM): iGiCiG, STOP2 (50 µM): iCiGiC, STOP Mix (50 µM): mixture of STOP1 and STOP2, synthesized by Metabion, Germany}, and 1 µl of T4 RNA Ligase (New England Biolabs, M0204S) for 16 hours at 16°C to ligate STOP oligos to the non-capped mRNA.

    Article Title: Strand-specific deep sequencing of the transcriptome
    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. .. After addition of 2× loading dye and incubation for 5 min at 65°C, the reaction was loaded onto a denaturing urea-PAGE gel in order to separate the fragments with ligated 3′ adapter from nonligated adapter (band sizes: insert size + 23 nt for single end sequencing; insert size + 34 nt for paired end libraries).

    other:

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

    Sequencing:

    Article Title: The unfolded protein response and endoplasmic reticulum protein targeting machineries converge on the stress sensor IRE1
    Article Snippet: .. Preparation of small RNA cDNA libraries for deep sequencing 1 μL of RA3 RNA 3’ adapter (5’-TGGAATTCTCGGGTGCCAAGG-3’) from the TruSeq RNA small Sample Prep Kit (Illumina) was added to each of the purified RNA samples above, and the mixture was placed at 80°C for 2 min, then placed immediately on ice for another 2 min. 1.5 μL of 10× T4 RNA ligase reaction buffer (500 mM Tris pH 7.5, 100 mM MgCl2 , 10 mM DTT; New England Biolabs) and 4.5 μL 50% PEG 8,000 (New England Biolabs), 1 μL RNase inhibitor (Illumina), and 1 μL (200 units) T4 RNA ligase 2, truncated R55K, K227Q (KQ) mutant (New England Biolabs) were added to each sample and the reactions were incubated at 16°C overnight. .. 12–16 hr later, 0.5 μL of 10× T4 RNA ligase reaction buffer, 1.5 μL of 50% PEG 8,000, 2 μL of RNase-free water and 1 μL (200 units) T4 RNA ligase 2 truncated KQ were added to each sample and the reactions were incubated an additional 2 hr at 25°C.

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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: 96/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rnl2 reaction buffer/product/New England Biolabs
    Average 96 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    t4 rnl2 reaction buffer - by Bioz Stars, 2020-05
    96/100 stars
      Buy from Supplier

    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 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase 2 truncated buffer/product/New England Biolabs
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase 2 truncated buffer - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    Image Search Results


    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

    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

    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