t4 pnk buffer  (New England Biolabs)


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    New England Biolabs t4 pnk buffer
    Endonucleolytically cleaved 5’-OH RNAs are phosphorylated by Trl1. a 8% PAGE followed by northern blot analysis using probe prA. Levels of 3’-NGD RNA fragments in trl1/dom34 cells compared with those from TRL1/dom34 cells. b B1 and B4 RNA quantification relative to 5S rRNA from three independent experiments as shown in ( a ). c 12% PAGE followed by northern blot analysis using probe prA. Treatment using <t>T4</t> PNK to determine 5’-OH and 5’-P B4 RNA positions in the indicated strains. One-fourth of trl1/dom34 total RNA treated was loaded to limit scan saturation and allow TRL1/dom34 B4 RNA detection. The 5S rRNA served as a loading control. d As in Fig. 3a , Xrn1 digestion of total RNA extracts from trl1/dom34 mutant cells in the presence or absence of T4 PNK treatment in vitro . A minor band detected in trl1 is indicated by an asterisk (see also Supplementary Fig. 5 in which this band is detectable in TRL1 cells). Error bars indicate standard deviation (s.d) calculated from three independent experiments. Source data are provided as a Source Data file.
    T4 Pnk 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
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    Images

    1) Product Images from "No-Go Decay mRNA cleavage in the ribosome exit tunnel produces 5’-OH ends phosphorylated by Trl1"

    Article Title: No-Go Decay mRNA cleavage in the ribosome exit tunnel produces 5’-OH ends phosphorylated by Trl1

    Journal: bioRxiv

    doi: 10.1101/465633

    Endonucleolytically cleaved 5’-OH RNAs are phosphorylated by Trl1. a 8% PAGE followed by northern blot analysis using probe prA. Levels of 3’-NGD RNA fragments in trl1/dom34 cells compared with those from TRL1/dom34 cells. b B1 and B4 RNA quantification relative to 5S rRNA from three independent experiments as shown in ( a ). c 12% PAGE followed by northern blot analysis using probe prA. Treatment using T4 PNK to determine 5’-OH and 5’-P B4 RNA positions in the indicated strains. One-fourth of trl1/dom34 total RNA treated was loaded to limit scan saturation and allow TRL1/dom34 B4 RNA detection. The 5S rRNA served as a loading control. d As in Fig. 3a , Xrn1 digestion of total RNA extracts from trl1/dom34 mutant cells in the presence or absence of T4 PNK treatment in vitro . A minor band detected in trl1 is indicated by an asterisk (see also Supplementary Fig. 5 in which this band is detectable in TRL1 cells). Error bars indicate standard deviation (s.d) calculated from three independent experiments. Source data are provided as a Source Data file.
    Figure Legend Snippet: Endonucleolytically cleaved 5’-OH RNAs are phosphorylated by Trl1. a 8% PAGE followed by northern blot analysis using probe prA. Levels of 3’-NGD RNA fragments in trl1/dom34 cells compared with those from TRL1/dom34 cells. b B1 and B4 RNA quantification relative to 5S rRNA from three independent experiments as shown in ( a ). c 12% PAGE followed by northern blot analysis using probe prA. Treatment using T4 PNK to determine 5’-OH and 5’-P B4 RNA positions in the indicated strains. One-fourth of trl1/dom34 total RNA treated was loaded to limit scan saturation and allow TRL1/dom34 B4 RNA detection. The 5S rRNA served as a loading control. d As in Fig. 3a , Xrn1 digestion of total RNA extracts from trl1/dom34 mutant cells in the presence or absence of T4 PNK treatment in vitro . A minor band detected in trl1 is indicated by an asterisk (see also Supplementary Fig. 5 in which this band is detectable in TRL1 cells). Error bars indicate standard deviation (s.d) calculated from three independent experiments. Source data are provided as a Source Data file.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Northern Blot, RNA Detection, Mutagenesis, In Vitro, Standard Deviation

    2) Product Images from "Mechanical properties of DNA-like polymers"

    Article Title: Mechanical properties of DNA-like polymers

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt808

    Example measurement of mechanical properties. ( A ) Cyclization time course for 207-bp DNA-like polymer 5 (pJ1744). DNA ligase-catalyzed cyclization reaction was performed at ∼22°C with 1 nM DNA restriction fragment, T4 DNA ligation buffer (50 mM Tris–HCl, pH 7.5, 10 mM MgCl 2 , 1 mM ATP, 10 mM dithiothreitol) and a final concentration of 100 U/ml T4 DNA ligase. Aliquots (10 µl) were removed at 1–15 min time points, quenched by addition of EDTA to 20 mM and then analyzed by electrophoresis through 5% native polyacrylamide gels in 0.5× TBE buffer (50 mM Tris base, 55 mM boric acid and 1 mM EDTA, pH 8.3). Gel lanes contains Invitrogen 100 bp DNA ladder (M), linear monomer without ligase (0) and increasing 1-min time points of the ligation reaction ( 1–15 ) showing the evolution of linear monomer ( M ), linear dimer ( D ), circular monomer ( C M ) and circular dimer ( C D ). Nearest molecular weight bands are indicated. ( B ) Cyclization kinetics analysis for 207-bp DNA-like polymer 5 (pJ1744). Fitting of data in (A) determines the J -factor, as previously described ( 30 ) (see also Supplementary Data S3 ). ( C ) WLC analysis for DNA-like polymer 5 . Fit of experimental J -factor data using the WLC model. ( D ). Monte Carlo estimation of uncertainty. Fit of simulated J -factor data based on (C) using the WLC model.
    Figure Legend Snippet: Example measurement of mechanical properties. ( A ) Cyclization time course for 207-bp DNA-like polymer 5 (pJ1744). DNA ligase-catalyzed cyclization reaction was performed at ∼22°C with 1 nM DNA restriction fragment, T4 DNA ligation buffer (50 mM Tris–HCl, pH 7.5, 10 mM MgCl 2 , 1 mM ATP, 10 mM dithiothreitol) and a final concentration of 100 U/ml T4 DNA ligase. Aliquots (10 µl) were removed at 1–15 min time points, quenched by addition of EDTA to 20 mM and then analyzed by electrophoresis through 5% native polyacrylamide gels in 0.5× TBE buffer (50 mM Tris base, 55 mM boric acid and 1 mM EDTA, pH 8.3). Gel lanes contains Invitrogen 100 bp DNA ladder (M), linear monomer without ligase (0) and increasing 1-min time points of the ligation reaction ( 1–15 ) showing the evolution of linear monomer ( M ), linear dimer ( D ), circular monomer ( C M ) and circular dimer ( C D ). Nearest molecular weight bands are indicated. ( B ) Cyclization kinetics analysis for 207-bp DNA-like polymer 5 (pJ1744). Fitting of data in (A) determines the J -factor, as previously described ( 30 ) (see also Supplementary Data S3 ). ( C ) WLC analysis for DNA-like polymer 5 . Fit of experimental J -factor data using the WLC model. ( D ). Monte Carlo estimation of uncertainty. Fit of simulated J -factor data based on (C) using the WLC model.

    Techniques Used: DNA Ligation, Concentration Assay, Electrophoresis, Ligation, Molecular Weight

    3) Product Images from "CPA-seq reveals small ncRNAs with methylated nucleosides and diverse termini"

    Article Title: CPA-seq reveals small ncRNAs with methylated nucleosides and diverse termini

    Journal: Cell Discovery

    doi: 10.1038/s41421-021-00265-2

    CPA-seq. a The workflow of sRNA library preparation for CPA-seq. Purified small RNAs are incubated in deacylation buffer to remove 3′-aminoacyl (3′-aa), treated with Cap-Clip to remove 5′ m 7 G and m 3 G caps, then treated with T4 PNK to convert 5′-OH to 5′-P, and to convert 3′-P and 3′-cP to 3′-OH, followed by treatment with a mix of AlkB and AlkB(D135S) to remove methylations in m 1 G, m 3 C, and m 1 A. The pretreated small RNAs were ligated with 3′ and 5′ adapters, reverse transcribed by TGIRT-III, and then PCR amplified for sequencing. b Northern blotting of RNA samples from HEK293T with/without treatment of deacylation buffer. c Cap-Clip treated synthetic 5′-m 7 G-RNA (31 nt) was ligated with a 5′-adapter (26 nt). d T4 PNK-treated synthetic 5′-OH RNA (27 nt) was ligated with a 5′-adapter (26 nt). e T4 PNK-treated synthetic 3′-P RNA (27 nt) was ligated with 3′-adapter (29 nt). f LC-MS/MS analysis showed that sequential treatments with deacylation buffer, Cap-Clip, T4 PNK, and AlkB mix (CPA) efficiently removed methylations in m 1 G, m 3 C, and m 1 A of small RNAs extracted from HEK293T cells ( n = 3).
    Figure Legend Snippet: CPA-seq. a The workflow of sRNA library preparation for CPA-seq. Purified small RNAs are incubated in deacylation buffer to remove 3′-aminoacyl (3′-aa), treated with Cap-Clip to remove 5′ m 7 G and m 3 G caps, then treated with T4 PNK to convert 5′-OH to 5′-P, and to convert 3′-P and 3′-cP to 3′-OH, followed by treatment with a mix of AlkB and AlkB(D135S) to remove methylations in m 1 G, m 3 C, and m 1 A. The pretreated small RNAs were ligated with 3′ and 5′ adapters, reverse transcribed by TGIRT-III, and then PCR amplified for sequencing. b Northern blotting of RNA samples from HEK293T with/without treatment of deacylation buffer. c Cap-Clip treated synthetic 5′-m 7 G-RNA (31 nt) was ligated with a 5′-adapter (26 nt). d T4 PNK-treated synthetic 5′-OH RNA (27 nt) was ligated with a 5′-adapter (26 nt). e T4 PNK-treated synthetic 3′-P RNA (27 nt) was ligated with 3′-adapter (29 nt). f LC-MS/MS analysis showed that sequential treatments with deacylation buffer, Cap-Clip, T4 PNK, and AlkB mix (CPA) efficiently removed methylations in m 1 G, m 3 C, and m 1 A of small RNAs extracted from HEK293T cells ( n = 3).

    Techniques Used: Purification, Incubation, Cross-linking Immunoprecipitation, Polymerase Chain Reaction, Amplification, Sequencing, Northern Blot, Liquid Chromatography with Mass Spectroscopy

    CPA-seq reveals sRNAs with diverse termini. a Distribution of different types of sRNAs extracted from HEK293T cells that we process with the full CPA-seq process or with various combinations of the Cap-Clip, T4 PNK, and AlkB mix enzymes ( n = 2). b The number of sRNA species revealed with different treatments ( n = 2). c Distribution of different types of sRNAs responsive to T4 PNK treatment (unique reads that were highly detected in CPA group, but lowly detected in CA group with the fold change > 30, n = 2). d Reads of sRNA responsive to T4 PNK treatment mapping to 5S, 18S, and 28S ribosomal RNAs have been combined to show detection of rsRNAs containing diverse termini (rsRNAs with RPM > 300 are shown in the structural map). e Reads of sRNA responsive to T4 PNK treatment mapping to cytosolic tsRNAs have been combined to show detection of tsRNAs containing diverse termini. f Reads of tsRNAs responsive to T4 PNK treatment. g Northern blotting of GluCTC 5′tsRNAs that are responsive to T4 PNK treatment.
    Figure Legend Snippet: CPA-seq reveals sRNAs with diverse termini. a Distribution of different types of sRNAs extracted from HEK293T cells that we process with the full CPA-seq process or with various combinations of the Cap-Clip, T4 PNK, and AlkB mix enzymes ( n = 2). b The number of sRNA species revealed with different treatments ( n = 2). c Distribution of different types of sRNAs responsive to T4 PNK treatment (unique reads that were highly detected in CPA group, but lowly detected in CA group with the fold change > 30, n = 2). d Reads of sRNA responsive to T4 PNK treatment mapping to 5S, 18S, and 28S ribosomal RNAs have been combined to show detection of rsRNAs containing diverse termini (rsRNAs with RPM > 300 are shown in the structural map). e Reads of sRNA responsive to T4 PNK treatment mapping to cytosolic tsRNAs have been combined to show detection of tsRNAs containing diverse termini. f Reads of tsRNAs responsive to T4 PNK treatment. g Northern blotting of GluCTC 5′tsRNAs that are responsive to T4 PNK treatment.

    Techniques Used: Cross-linking Immunoprecipitation, Northern Blot

    4) Product Images from "Detection of circulating extracellular mRNAs by modified small-RNA-sequencing analysis"

    Article Title: Detection of circulating extracellular mRNAs by modified small-RNA-sequencing analysis

    Journal: JCI Insight

    doi: 10.1172/jci.insight.127317

    Read distribution of ex‑mRNA reads across the full-length mRNA transcripts. ( A and B ) Read coverage for the hemoglobin A2 transcript ( A ) and the albumin transcript ( B ) by sample type for untreated and T4 PNK end-treated samples. Exon boundaries (HBA2: 3 exons, ALB: 15 exons) are indicated by alternating intensities of gray, and UTRs are distinguished from CDS by thinner bars. ( C ) Metagene analysis with relative read coverage (percentage) across 5′ UTRs, CDSs, and 3′ UTRs for untreated and PNK-treated samples as well as corresponding data obtained after 100 random simulations (across an average of 2342–3500 captured transcripts for untreated samples and an average of 12,789–16,487 captured transcripts for PNK-treated samples, depending on sample type). Shown are results from n = 6 individual samples per condition.
    Figure Legend Snippet: Read distribution of ex‑mRNA reads across the full-length mRNA transcripts. ( A and B ) Read coverage for the hemoglobin A2 transcript ( A ) and the albumin transcript ( B ) by sample type for untreated and T4 PNK end-treated samples. Exon boundaries (HBA2: 3 exons, ALB: 15 exons) are indicated by alternating intensities of gray, and UTRs are distinguished from CDS by thinner bars. ( C ) Metagene analysis with relative read coverage (percentage) across 5′ UTRs, CDSs, and 3′ UTRs for untreated and PNK-treated samples as well as corresponding data obtained after 100 random simulations (across an average of 2342–3500 captured transcripts for untreated samples and an average of 12,789–16,487 captured transcripts for PNK-treated samples, depending on sample type). Shown are results from n = 6 individual samples per condition.

    Techniques Used:

    Treatment of total extracellular RNA with T4 polynucleotide kinase followed by small-RNA-sequencing. ( A ) Total RNA was isolated from 450 μl serum or platelet-depleted EDTA, acid citrate dextrose (ACD), and heparin plasma from 6 healthy individuals and purified using silica-based spin columns. Half of the RNA was treated with T4 polynucleotide kinase (T4 PNK) and repurified (PNK treated), and multiplexed small-RNA-sequencing (sRNA-seq) libraries were prepared separately for the untreated (libraries 1 and 3) and PNK-treated RNA (libraries 2 and 4). ( B ) Differences in read annotation in the 4 sample types for untreated RNA and PNK-treated RNA using initial annotation settings (reads 12–42 nt, up to 2 mismatches, multimapping). ( C ) Differences in ex‑mRNA capture between untreated and PNK-treated RNA using final annotation criteria (reads  > 15 nt, no mismatch and up to 2 mapping locations). Box plots show the median and first and third quartiles (bottom and top hinges). Whiskers extend at most ×1.5 interquartile range from the hinges; any data outside this are shown as individual outlier points. Shown are results from  n  = 6 individual samples per condition.
    Figure Legend Snippet: Treatment of total extracellular RNA with T4 polynucleotide kinase followed by small-RNA-sequencing. ( A ) Total RNA was isolated from 450 μl serum or platelet-depleted EDTA, acid citrate dextrose (ACD), and heparin plasma from 6 healthy individuals and purified using silica-based spin columns. Half of the RNA was treated with T4 polynucleotide kinase (T4 PNK) and repurified (PNK treated), and multiplexed small-RNA-sequencing (sRNA-seq) libraries were prepared separately for the untreated (libraries 1 and 3) and PNK-treated RNA (libraries 2 and 4). ( B ) Differences in read annotation in the 4 sample types for untreated RNA and PNK-treated RNA using initial annotation settings (reads 12–42 nt, up to 2 mismatches, multimapping). ( C ) Differences in ex‑mRNA capture between untreated and PNK-treated RNA using final annotation criteria (reads > 15 nt, no mismatch and up to 2 mapping locations). Box plots show the median and first and third quartiles (bottom and top hinges). Whiskers extend at most ×1.5 interquartile range from the hinges; any data outside this are shown as individual outlier points. Shown are results from n = 6 individual samples per condition.

    Techniques Used: RNA Sequencing Assay, Isolation, Purification

    5) Product Images from "Comprehensive analysis of the Corynebacterium glutamicum transcriptome using an improved RNAseq technique"

    Article Title: Comprehensive analysis of the Corynebacterium glutamicum transcriptome using an improved RNAseq technique

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-14-888

    Experimental workflow for the preparation of a whole transcriptome library (a) and of a library enriched for primary 5′-transcript ends (b). Both protocols start with isolated total RNA. Stable RNA is then depleted using the Ribo-Zero rRNA removal kit and the obtained RNA is fragmented my metal hydrolysis to a size of 200 - 500 nt. For the whole transcriptome library (a) the 5′-triphosphate ends are processed to 5′-monophosphate ends by a RNA 5′-polyphosphatase, unphosphorylated 5′-ends are phosphorylated, and phosphorylated 3′-ends are then dephosphorylated using T4 polynucleotide kinase. For the native 5′-end protocol (b) , all fragments containing a 5′-monophosphate are degraded by treatment with a 5′-phosphate dependent exonuclease and the 5′-triphosphate ends of native transcripts are then processed to 5′-monophosphate ends by a RNA 5′-polyphosphatase. Next, for both libraries RNA adapters are ligated to the 5′-ends carrying a 5′-monophosphate group. The tagging of the 3′-end of the RNA with flanking sequences necessary for reverse transcription is performed in a ligation-free approach with a loop DNA adapter containing seven unpaired wobble bases at its 3′-end. After reverse transcription of the RNA fragments into cDNA fragments, the cDNA fragments are amplified, tagged with sequencing linkers at their ends by PCR and finally sequenced. Stable RNA species (rRNA, tRNA) are depicted in red, other RNAs are given in green, and DNA in blue.
    Figure Legend Snippet: Experimental workflow for the preparation of a whole transcriptome library (a) and of a library enriched for primary 5′-transcript ends (b). Both protocols start with isolated total RNA. Stable RNA is then depleted using the Ribo-Zero rRNA removal kit and the obtained RNA is fragmented my metal hydrolysis to a size of 200 - 500 nt. For the whole transcriptome library (a) the 5′-triphosphate ends are processed to 5′-monophosphate ends by a RNA 5′-polyphosphatase, unphosphorylated 5′-ends are phosphorylated, and phosphorylated 3′-ends are then dephosphorylated using T4 polynucleotide kinase. For the native 5′-end protocol (b) , all fragments containing a 5′-monophosphate are degraded by treatment with a 5′-phosphate dependent exonuclease and the 5′-triphosphate ends of native transcripts are then processed to 5′-monophosphate ends by a RNA 5′-polyphosphatase. Next, for both libraries RNA adapters are ligated to the 5′-ends carrying a 5′-monophosphate group. The tagging of the 3′-end of the RNA with flanking sequences necessary for reverse transcription is performed in a ligation-free approach with a loop DNA adapter containing seven unpaired wobble bases at its 3′-end. After reverse transcription of the RNA fragments into cDNA fragments, the cDNA fragments are amplified, tagged with sequencing linkers at their ends by PCR and finally sequenced. Stable RNA species (rRNA, tRNA) are depicted in red, other RNAs are given in green, and DNA in blue.

    Techniques Used: Isolation, Ligation, Amplification, Sequencing, Polymerase Chain Reaction

    6) Product Images from "Nascent RNA sequencing reveals distinct features in plant transcription"

    Article Title: Nascent RNA sequencing reveals distinct features in plant transcription

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

    doi: 10.1073/pnas.1603217113

    ( A ) Effect of enzymes on 5′ monophosporylated (5′Pi) or capped RNA (CAP). T4 RNAP synthesized RNA (264 nt) was kinased using T4 PNK and [α- 32 P]ATP or capped with the Vaccinia Capping System (M2080) and [α- 32 P]GTP, as described by the manufacturer. ( B ) Comparison of RppH activity on 32 P-capped RNA in buffer NEB II vs. NEB T4 RNA ligase buffer. ( C ) 32 P-capped RNA (10 pmol) (264 nt) incubated with 0.5 U of RppH at 37 °C and 20 °C. ( D ) [ 32 P]5′-adenylated oligo (20 pmol) (55 nt) incubated with 2 U of RppH at 20 °C and 37 °C in T4 RNA ligase buffer. ( E ) Assessment of run-on length: nuclei were run on using the described run-on conditions (20 nM CTP-limiting) for the indicated time in the presence and absence of 4 ng/µL α-amanitin, a concentration efficiently inhibiting RNAP II transcription. For visualization of actual run-on length, nuclei were incubated in Freezing Buffer + RNase A (0.25 mg/mL) for 20 min at 4 °C followed by 5 min at RT and consecutively washed three times before run-on.
    Figure Legend Snippet: ( A ) Effect of enzymes on 5′ monophosporylated (5′Pi) or capped RNA (CAP). T4 RNAP synthesized RNA (264 nt) was kinased using T4 PNK and [α- 32 P]ATP or capped with the Vaccinia Capping System (M2080) and [α- 32 P]GTP, as described by the manufacturer. ( B ) Comparison of RppH activity on 32 P-capped RNA in buffer NEB II vs. NEB T4 RNA ligase buffer. ( C ) 32 P-capped RNA (10 pmol) (264 nt) incubated with 0.5 U of RppH at 37 °C and 20 °C. ( D ) [ 32 P]5′-adenylated oligo (20 pmol) (55 nt) incubated with 2 U of RppH at 20 °C and 37 °C in T4 RNA ligase buffer. ( E ) Assessment of run-on length: nuclei were run on using the described run-on conditions (20 nM CTP-limiting) for the indicated time in the presence and absence of 4 ng/µL α-amanitin, a concentration efficiently inhibiting RNAP II transcription. For visualization of actual run-on length, nuclei were incubated in Freezing Buffer + RNase A (0.25 mg/mL) for 20 min at 4 °C followed by 5 min at RT and consecutively washed three times before run-on.

    Techniques Used: Synthesized, Activity Assay, Incubation, Concentration Assay

    7) Product Images from "A Simple and Cost-Effective Approach for In Vitro Production of Sliced siRNAs as Potent Triggers for RNAi"

    Article Title: A Simple and Cost-Effective Approach for In Vitro Production of Sliced siRNAs as Potent Triggers for RNAi

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2017.07.008

    Manipulation of 5′ppp-Triggered Interferon Response HEK293 cells were transfected with poly(I:C) or several tsli-siRNAs. The final concentration of 10 nM for each RNAi reagent was used in transfection for qPCR assay. Gene expression level changes in OAS1, IRF9, CDKL, and IFNB relative to GAPDH were measured by qPCR. (A) Mild interferon response was observed from all four tsli-siRNAs, with tsli-RRM2 having the strongest response among them. G-tsli-Stat3 exhibited a much stronger response than tsli-Stat3, and GG-tsli-Stat3 reversed this effect to some extent. (B) CIP treatment minimized the strong interferon response by G-tsli-Stat3. (C) CIP treatment minimized and T4 PNK treatment elevated the interferon response by tsli-RRM2. Fold changes in gene expression were normalized to untreated HEK293 cells. Details of qPCR procedure and results calculation were provided in the Materials and Methods . Error bars indicate SD.
    Figure Legend Snippet: Manipulation of 5′ppp-Triggered Interferon Response HEK293 cells were transfected with poly(I:C) or several tsli-siRNAs. The final concentration of 10 nM for each RNAi reagent was used in transfection for qPCR assay. Gene expression level changes in OAS1, IRF9, CDKL, and IFNB relative to GAPDH were measured by qPCR. (A) Mild interferon response was observed from all four tsli-siRNAs, with tsli-RRM2 having the strongest response among them. G-tsli-Stat3 exhibited a much stronger response than tsli-Stat3, and GG-tsli-Stat3 reversed this effect to some extent. (B) CIP treatment minimized the strong interferon response by G-tsli-Stat3. (C) CIP treatment minimized and T4 PNK treatment elevated the interferon response by tsli-RRM2. Fold changes in gene expression were normalized to untreated HEK293 cells. Details of qPCR procedure and results calculation were provided in the Materials and Methods . Error bars indicate SD.

    Techniques Used: Transfection, Concentration Assay, Real-time Polymerase Chain Reaction, Expressing

    8) 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

    Post-digest Labeling Reaction Scheme (A) The γ-phosphate of 18 O 4 -γ-ATP is transferred to the 5′-hydroxy group of a cross-linked RNA oligonucleotide. T4-PNK also catalyzes a 3′-dephosphorylation reaction. (B) Simulation of the 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: Post-digest Labeling Reaction Scheme (A) The γ-phosphate of 18 O 4 -γ-ATP is transferred to the 5′-hydroxy group of a cross-linked RNA oligonucleotide. T4-PNK also catalyzes a 3′-dephosphorylation reaction. (B) Simulation of the 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, De-Phosphorylation Assay

    9) Product Images from "No-Go Decay mRNA cleavage in the ribosome exit tunnel produces 5′-OH ends phosphorylated by Trl1"

    Article Title: No-Go Decay mRNA cleavage in the ribosome exit tunnel produces 5′-OH ends phosphorylated by Trl1

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13991-9

    Endonucleolytically cleaved 5′-OH RNAs are phosphorylated by Trl1. a 8% PAGE followed by northern blot analysis using probe prA. Levels of 3′-NGD RNA fragments in trl1/dom34 cells compared with those from TRL1/dom34 cells. b B1 and B4 RNA quantification relative to 5S rRNA from three independent experiments as shown in a . c 12% PAGE followed by northern blot analysis using probe prA. Treatment using T4 PNK to determine 5’-OH and 5’-P B4 RNA positions in the indicated strains. One-fourth of trl1/dom34 total RNA treated was loaded to limit scan saturation and allow TRL1/dom34 B4 RNA detection. The 5S rRNA served as a loading control. d As in Fig. 3a , Xrn1 digestion of total RNA extracts from trl1/dom34 mutant cells in the presence or absence of T4 PNK treatment in vitro. A minor band detected in trl1 is indicated by an asterisk (see also Supplementary Fig. 5 in which this band is detectable in TRL1 cells). Error bars indicate standard deviation (s.d.) calculated from three independent experiments. Source data are provided as a Source Data file.
    Figure Legend Snippet: Endonucleolytically cleaved 5′-OH RNAs are phosphorylated by Trl1. a 8% PAGE followed by northern blot analysis using probe prA. Levels of 3′-NGD RNA fragments in trl1/dom34 cells compared with those from TRL1/dom34 cells. b B1 and B4 RNA quantification relative to 5S rRNA from three independent experiments as shown in a . c 12% PAGE followed by northern blot analysis using probe prA. Treatment using T4 PNK to determine 5’-OH and 5’-P B4 RNA positions in the indicated strains. One-fourth of trl1/dom34 total RNA treated was loaded to limit scan saturation and allow TRL1/dom34 B4 RNA detection. The 5S rRNA served as a loading control. d As in Fig. 3a , Xrn1 digestion of total RNA extracts from trl1/dom34 mutant cells in the presence or absence of T4 PNK treatment in vitro. A minor band detected in trl1 is indicated by an asterisk (see also Supplementary Fig. 5 in which this band is detectable in TRL1 cells). Error bars indicate standard deviation (s.d.) calculated from three independent experiments. Source data are provided as a Source Data file.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Northern Blot, RNA Detection, Mutagenesis, In Vitro, Standard Deviation

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    New England Biolabs t4 pnk buffer
    Endonucleolytically cleaved 5’-OH RNAs are phosphorylated by Trl1. a 8% PAGE followed by northern blot analysis using probe prA. Levels of 3’-NGD RNA fragments in trl1/dom34 cells compared with those from TRL1/dom34 cells. b B1 and B4 RNA quantification relative to 5S rRNA from three independent experiments as shown in ( a ). c 12% PAGE followed by northern blot analysis using probe prA. Treatment using <t>T4</t> PNK to determine 5’-OH and 5’-P B4 RNA positions in the indicated strains. One-fourth of trl1/dom34 total RNA treated was loaded to limit scan saturation and allow TRL1/dom34 B4 RNA detection. The 5S rRNA served as a loading control. d As in Fig. 3a , Xrn1 digestion of total RNA extracts from trl1/dom34 mutant cells in the presence or absence of T4 PNK treatment in vitro . A minor band detected in trl1 is indicated by an asterisk (see also Supplementary Fig. 5 in which this band is detectable in TRL1 cells). Error bars indicate standard deviation (s.d) calculated from three independent experiments. Source data are provided as a Source Data file.
    T4 Pnk 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
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    Endonucleolytically cleaved 5’-OH RNAs are phosphorylated by Trl1. a 8% PAGE followed by northern blot analysis using probe prA. Levels of 3’-NGD RNA fragments in trl1/dom34 cells compared with those from TRL1/dom34 cells. b B1 and B4 RNA quantification relative to 5S rRNA from three independent experiments as shown in ( a ). c 12% PAGE followed by northern blot analysis using probe prA. Treatment using T4 PNK to determine 5’-OH and 5’-P B4 RNA positions in the indicated strains. One-fourth of trl1/dom34 total RNA treated was loaded to limit scan saturation and allow TRL1/dom34 B4 RNA detection. The 5S rRNA served as a loading control. d As in Fig. 3a , Xrn1 digestion of total RNA extracts from trl1/dom34 mutant cells in the presence or absence of T4 PNK treatment in vitro . A minor band detected in trl1 is indicated by an asterisk (see also Supplementary Fig. 5 in which this band is detectable in TRL1 cells). Error bars indicate standard deviation (s.d) calculated from three independent experiments. Source data are provided as a Source Data file.

    Journal: bioRxiv

    Article Title: No-Go Decay mRNA cleavage in the ribosome exit tunnel produces 5’-OH ends phosphorylated by Trl1

    doi: 10.1101/465633

    Figure Lengend Snippet: Endonucleolytically cleaved 5’-OH RNAs are phosphorylated by Trl1. a 8% PAGE followed by northern blot analysis using probe prA. Levels of 3’-NGD RNA fragments in trl1/dom34 cells compared with those from TRL1/dom34 cells. b B1 and B4 RNA quantification relative to 5S rRNA from three independent experiments as shown in ( a ). c 12% PAGE followed by northern blot analysis using probe prA. Treatment using T4 PNK to determine 5’-OH and 5’-P B4 RNA positions in the indicated strains. One-fourth of trl1/dom34 total RNA treated was loaded to limit scan saturation and allow TRL1/dom34 B4 RNA detection. The 5S rRNA served as a loading control. d As in Fig. 3a , Xrn1 digestion of total RNA extracts from trl1/dom34 mutant cells in the presence or absence of T4 PNK treatment in vitro . A minor band detected in trl1 is indicated by an asterisk (see also Supplementary Fig. 5 in which this band is detectable in TRL1 cells). Error bars indicate standard deviation (s.d) calculated from three independent experiments. Source data are provided as a Source Data file.

    Article Snippet: NEB Buffer 3 was replaced by T4 PNK buffer (NEB) in kinase assays in the presence or absence of Xrn1 (Figs and ).

    Techniques: Polyacrylamide Gel Electrophoresis, Northern Blot, RNA Detection, Mutagenesis, In Vitro, Standard Deviation

    Example measurement of mechanical properties. ( A ) Cyclization time course for 207-bp DNA-like polymer 5 (pJ1744). DNA ligase-catalyzed cyclization reaction was performed at ∼22°C with 1 nM DNA restriction fragment, T4 DNA ligation buffer (50 mM Tris–HCl, pH 7.5, 10 mM MgCl 2 , 1 mM ATP, 10 mM dithiothreitol) and a final concentration of 100 U/ml T4 DNA ligase. Aliquots (10 µl) were removed at 1–15 min time points, quenched by addition of EDTA to 20 mM and then analyzed by electrophoresis through 5% native polyacrylamide gels in 0.5× TBE buffer (50 mM Tris base, 55 mM boric acid and 1 mM EDTA, pH 8.3). Gel lanes contains Invitrogen 100 bp DNA ladder (M), linear monomer without ligase (0) and increasing 1-min time points of the ligation reaction ( 1–15 ) showing the evolution of linear monomer ( M ), linear dimer ( D ), circular monomer ( C M ) and circular dimer ( C D ). Nearest molecular weight bands are indicated. ( B ) Cyclization kinetics analysis for 207-bp DNA-like polymer 5 (pJ1744). Fitting of data in (A) determines the J -factor, as previously described ( 30 ) (see also Supplementary Data S3 ). ( C ) WLC analysis for DNA-like polymer 5 . Fit of experimental J -factor data using the WLC model. ( D ). Monte Carlo estimation of uncertainty. Fit of simulated J -factor data based on (C) using the WLC model.

    Journal: Nucleic Acids Research

    Article Title: Mechanical properties of DNA-like polymers

    doi: 10.1093/nar/gkt808

    Figure Lengend Snippet: Example measurement of mechanical properties. ( A ) Cyclization time course for 207-bp DNA-like polymer 5 (pJ1744). DNA ligase-catalyzed cyclization reaction was performed at ∼22°C with 1 nM DNA restriction fragment, T4 DNA ligation buffer (50 mM Tris–HCl, pH 7.5, 10 mM MgCl 2 , 1 mM ATP, 10 mM dithiothreitol) and a final concentration of 100 U/ml T4 DNA ligase. Aliquots (10 µl) were removed at 1–15 min time points, quenched by addition of EDTA to 20 mM and then analyzed by electrophoresis through 5% native polyacrylamide gels in 0.5× TBE buffer (50 mM Tris base, 55 mM boric acid and 1 mM EDTA, pH 8.3). Gel lanes contains Invitrogen 100 bp DNA ladder (M), linear monomer without ligase (0) and increasing 1-min time points of the ligation reaction ( 1–15 ) showing the evolution of linear monomer ( M ), linear dimer ( D ), circular monomer ( C M ) and circular dimer ( C D ). Nearest molecular weight bands are indicated. ( B ) Cyclization kinetics analysis for 207-bp DNA-like polymer 5 (pJ1744). Fitting of data in (A) determines the J -factor, as previously described ( 30 ) (see also Supplementary Data S3 ). ( C ) WLC analysis for DNA-like polymer 5 . Fit of experimental J -factor data using the WLC model. ( D ). Monte Carlo estimation of uncertainty. Fit of simulated J -factor data based on (C) using the WLC model.

    Article Snippet: DNA ligase-catalyzed cyclization reactions were performed at ∼22°C with 1 nM DNA restriction fragment, T4 DNA ligation buffer (50 mM Tris–HCl, pH 7.5, 10 mM MgCl2 , 1 mM ATP, 10 mM dithiothreitol; NEB) and a final concentration of 100 U/ml T4 DNA ligase (NEB).

    Techniques: DNA Ligation, Concentration Assay, Electrophoresis, Ligation, Molecular Weight

    CPA-seq. a The workflow of sRNA library preparation for CPA-seq. Purified small RNAs are incubated in deacylation buffer to remove 3′-aminoacyl (3′-aa), treated with Cap-Clip to remove 5′ m 7 G and m 3 G caps, then treated with T4 PNK to convert 5′-OH to 5′-P, and to convert 3′-P and 3′-cP to 3′-OH, followed by treatment with a mix of AlkB and AlkB(D135S) to remove methylations in m 1 G, m 3 C, and m 1 A. The pretreated small RNAs were ligated with 3′ and 5′ adapters, reverse transcribed by TGIRT-III, and then PCR amplified for sequencing. b Northern blotting of RNA samples from HEK293T with/without treatment of deacylation buffer. c Cap-Clip treated synthetic 5′-m 7 G-RNA (31 nt) was ligated with a 5′-adapter (26 nt). d T4 PNK-treated synthetic 5′-OH RNA (27 nt) was ligated with a 5′-adapter (26 nt). e T4 PNK-treated synthetic 3′-P RNA (27 nt) was ligated with 3′-adapter (29 nt). f LC-MS/MS analysis showed that sequential treatments with deacylation buffer, Cap-Clip, T4 PNK, and AlkB mix (CPA) efficiently removed methylations in m 1 G, m 3 C, and m 1 A of small RNAs extracted from HEK293T cells ( n = 3).

    Journal: Cell Discovery

    Article Title: CPA-seq reveals small ncRNAs with methylated nucleosides and diverse termini

    doi: 10.1038/s41421-021-00265-2

    Figure Lengend Snippet: CPA-seq. a The workflow of sRNA library preparation for CPA-seq. Purified small RNAs are incubated in deacylation buffer to remove 3′-aminoacyl (3′-aa), treated with Cap-Clip to remove 5′ m 7 G and m 3 G caps, then treated with T4 PNK to convert 5′-OH to 5′-P, and to convert 3′-P and 3′-cP to 3′-OH, followed by treatment with a mix of AlkB and AlkB(D135S) to remove methylations in m 1 G, m 3 C, and m 1 A. The pretreated small RNAs were ligated with 3′ and 5′ adapters, reverse transcribed by TGIRT-III, and then PCR amplified for sequencing. b Northern blotting of RNA samples from HEK293T with/without treatment of deacylation buffer. c Cap-Clip treated synthetic 5′-m 7 G-RNA (31 nt) was ligated with a 5′-adapter (26 nt). d T4 PNK-treated synthetic 5′-OH RNA (27 nt) was ligated with a 5′-adapter (26 nt). e T4 PNK-treated synthetic 3′-P RNA (27 nt) was ligated with 3′-adapter (29 nt). f LC-MS/MS analysis showed that sequential treatments with deacylation buffer, Cap-Clip, T4 PNK, and AlkB mix (CPA) efficiently removed methylations in m 1 G, m 3 C, and m 1 A of small RNAs extracted from HEK293T cells ( n = 3).

    Article Snippet: Then the recovered small RNA was incubated with 1 U Cap-Clip Acid Pyrophosphatase (Cellscript) in 1× Cap-Clip Acid Pyrophosphatase reaction buffer (Cellscript) at 37 °C for 30 min. Then, the reaction was added with 20 U T4 PNK (NEB) in 1× T4 PNK reaction buffer (NEB) and 1 mM ATP (NEB) and incubated at 37 °C for 30 min.

    Techniques: Purification, Incubation, Cross-linking Immunoprecipitation, Polymerase Chain Reaction, Amplification, Sequencing, Northern Blot, Liquid Chromatography with Mass Spectroscopy

    CPA-seq reveals sRNAs with diverse termini. a Distribution of different types of sRNAs extracted from HEK293T cells that we process with the full CPA-seq process or with various combinations of the Cap-Clip, T4 PNK, and AlkB mix enzymes ( n = 2). b The number of sRNA species revealed with different treatments ( n = 2). c Distribution of different types of sRNAs responsive to T4 PNK treatment (unique reads that were highly detected in CPA group, but lowly detected in CA group with the fold change > 30, n = 2). d Reads of sRNA responsive to T4 PNK treatment mapping to 5S, 18S, and 28S ribosomal RNAs have been combined to show detection of rsRNAs containing diverse termini (rsRNAs with RPM > 300 are shown in the structural map). e Reads of sRNA responsive to T4 PNK treatment mapping to cytosolic tsRNAs have been combined to show detection of tsRNAs containing diverse termini. f Reads of tsRNAs responsive to T4 PNK treatment. g Northern blotting of GluCTC 5′tsRNAs that are responsive to T4 PNK treatment.

    Journal: Cell Discovery

    Article Title: CPA-seq reveals small ncRNAs with methylated nucleosides and diverse termini

    doi: 10.1038/s41421-021-00265-2

    Figure Lengend Snippet: CPA-seq reveals sRNAs with diverse termini. a Distribution of different types of sRNAs extracted from HEK293T cells that we process with the full CPA-seq process or with various combinations of the Cap-Clip, T4 PNK, and AlkB mix enzymes ( n = 2). b The number of sRNA species revealed with different treatments ( n = 2). c Distribution of different types of sRNAs responsive to T4 PNK treatment (unique reads that were highly detected in CPA group, but lowly detected in CA group with the fold change > 30, n = 2). d Reads of sRNA responsive to T4 PNK treatment mapping to 5S, 18S, and 28S ribosomal RNAs have been combined to show detection of rsRNAs containing diverse termini (rsRNAs with RPM > 300 are shown in the structural map). e Reads of sRNA responsive to T4 PNK treatment mapping to cytosolic tsRNAs have been combined to show detection of tsRNAs containing diverse termini. f Reads of tsRNAs responsive to T4 PNK treatment. g Northern blotting of GluCTC 5′tsRNAs that are responsive to T4 PNK treatment.

    Article Snippet: Then the recovered small RNA was incubated with 1 U Cap-Clip Acid Pyrophosphatase (Cellscript) in 1× Cap-Clip Acid Pyrophosphatase reaction buffer (Cellscript) at 37 °C for 30 min. Then, the reaction was added with 20 U T4 PNK (NEB) in 1× T4 PNK reaction buffer (NEB) and 1 mM ATP (NEB) and incubated at 37 °C for 30 min.

    Techniques: Cross-linking Immunoprecipitation, Northern Blot

    Read distribution of ex‑mRNA reads across the full-length mRNA transcripts. ( A and B ) Read coverage for the hemoglobin A2 transcript ( A ) and the albumin transcript ( B ) by sample type for untreated and T4 PNK end-treated samples. Exon boundaries (HBA2: 3 exons, ALB: 15 exons) are indicated by alternating intensities of gray, and UTRs are distinguished from CDS by thinner bars. ( C ) Metagene analysis with relative read coverage (percentage) across 5′ UTRs, CDSs, and 3′ UTRs for untreated and PNK-treated samples as well as corresponding data obtained after 100 random simulations (across an average of 2342–3500 captured transcripts for untreated samples and an average of 12,789–16,487 captured transcripts for PNK-treated samples, depending on sample type). Shown are results from n = 6 individual samples per condition.

    Journal: JCI Insight

    Article Title: Detection of circulating extracellular mRNAs by modified small-RNA-sequencing analysis

    doi: 10.1172/jci.insight.127317

    Figure Lengend Snippet: Read distribution of ex‑mRNA reads across the full-length mRNA transcripts. ( A and B ) Read coverage for the hemoglobin A2 transcript ( A ) and the albumin transcript ( B ) by sample type for untreated and T4 PNK end-treated samples. Exon boundaries (HBA2: 3 exons, ALB: 15 exons) are indicated by alternating intensities of gray, and UTRs are distinguished from CDS by thinner bars. ( C ) Metagene analysis with relative read coverage (percentage) across 5′ UTRs, CDSs, and 3′ UTRs for untreated and PNK-treated samples as well as corresponding data obtained after 100 random simulations (across an average of 2342–3500 captured transcripts for untreated samples and an average of 12,789–16,487 captured transcripts for PNK-treated samples, depending on sample type). Shown are results from n = 6 individual samples per condition.

    Article Snippet: To half of the eluted exRNA, i.e., 14 μl, we added 6 μl of a master mix corresponding to the equivalent of 2 μl ×10 T4 PNK buffer, 2 μl 10 mM ATP, 1 μl RNase-free water, and 1 μl T4 PNK (NEB, catalog M0201S) for a final reaction volume of 20 μl in a 1.5 ml siliconized microcentrifuge tube.

    Techniques:

    Treatment of total extracellular RNA with T4 polynucleotide kinase followed by small-RNA-sequencing. ( A ) Total RNA was isolated from 450 μl serum or platelet-depleted EDTA, acid citrate dextrose (ACD), and heparin plasma from 6 healthy individuals and purified using silica-based spin columns. Half of the RNA was treated with T4 polynucleotide kinase (T4 PNK) and repurified (PNK treated), and multiplexed small-RNA-sequencing (sRNA-seq) libraries were prepared separately for the untreated (libraries 1 and 3) and PNK-treated RNA (libraries 2 and 4). ( B ) Differences in read annotation in the 4 sample types for untreated RNA and PNK-treated RNA using initial annotation settings (reads 12–42 nt, up to 2 mismatches, multimapping). ( C ) Differences in ex‑mRNA capture between untreated and PNK-treated RNA using final annotation criteria (reads  > 15 nt, no mismatch and up to 2 mapping locations). Box plots show the median and first and third quartiles (bottom and top hinges). Whiskers extend at most ×1.5 interquartile range from the hinges; any data outside this are shown as individual outlier points. Shown are results from  n  = 6 individual samples per condition.

    Journal: JCI Insight

    Article Title: Detection of circulating extracellular mRNAs by modified small-RNA-sequencing analysis

    doi: 10.1172/jci.insight.127317

    Figure Lengend Snippet: Treatment of total extracellular RNA with T4 polynucleotide kinase followed by small-RNA-sequencing. ( A ) Total RNA was isolated from 450 μl serum or platelet-depleted EDTA, acid citrate dextrose (ACD), and heparin plasma from 6 healthy individuals and purified using silica-based spin columns. Half of the RNA was treated with T4 polynucleotide kinase (T4 PNK) and repurified (PNK treated), and multiplexed small-RNA-sequencing (sRNA-seq) libraries were prepared separately for the untreated (libraries 1 and 3) and PNK-treated RNA (libraries 2 and 4). ( B ) Differences in read annotation in the 4 sample types for untreated RNA and PNK-treated RNA using initial annotation settings (reads 12–42 nt, up to 2 mismatches, multimapping). ( C ) Differences in ex‑mRNA capture between untreated and PNK-treated RNA using final annotation criteria (reads > 15 nt, no mismatch and up to 2 mapping locations). Box plots show the median and first and third quartiles (bottom and top hinges). Whiskers extend at most ×1.5 interquartile range from the hinges; any data outside this are shown as individual outlier points. Shown are results from n = 6 individual samples per condition.

    Article Snippet: To half of the eluted exRNA, i.e., 14 μl, we added 6 μl of a master mix corresponding to the equivalent of 2 μl ×10 T4 PNK buffer, 2 μl 10 mM ATP, 1 μl RNase-free water, and 1 μl T4 PNK (NEB, catalog M0201S) for a final reaction volume of 20 μl in a 1.5 ml siliconized microcentrifuge tube.

    Techniques: RNA Sequencing Assay, Isolation, Purification