t4 pnk  (New England Biolabs)


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    T4 Polynucleotide Kinase
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    T4 Polynucleotide Kinase 2 500 units
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    M0201L
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    Category:
    Polynucleotide Kinases
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    2 500 units
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    New England Biolabs t4 pnk
    T4 Polynucleotide Kinase
    T4 Polynucleotide Kinase 2 500 units
    https://www.bioz.com/result/t4 pnk/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    t4 pnk - by Bioz Stars, 2021-06
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    Images

    1) Product Images from "Tagsteady: a metabarcoding library preparation protocol to avoid false assignment of sequences to samples"

    Article Title: Tagsteady: a metabarcoding library preparation protocol to avoid false assignment of sequences to samples

    Journal: bioRxiv

    doi: 10.1101/2020.01.22.915009

    Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, T4 PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).
    Figure Legend Snippet: Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, T4 PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).

    Techniques Used: Polymerase Chain Reaction, Activity Assay, Sequencing, Ligation, Amplification

    2) Product Images from "Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities"

    Article Title: Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00484-w

    yUsb1 acts as a 3′–5′exonuclease and CPDase in vitro. a U6 snRNA is synthesized by RNA Polymerase III. Transcription termination produces a heterogeneous U6 with a 4–8 nucleotide U-tail. Processing by yUsb1 shortens the U-tail and leaves a phosphoryl group. b Usb1 removes nucleotides from the 3′ end of RNAs. The 5′-labeled U6 95–112+3U oligonucleotide cis -diol substrate (lane 2) is insensitive to CIP (lane 3) or T4 PNK (lane 4) treatment. Incubation with yUsb1 for 1 h results in a shorter product (lane 5). Similar reactivity of the product to both CIP (lane 6) and T4 PNK (lane 7) indicates that the product is a noncyclic phosphate. An alkaline hydrolysis ladder (lane 1) shows the mobility of oligonucleotide products of different lengths. ( c , top ) One-dimensional 31 P NMR spectra of 2′,3′-cUMP shows a single peak at 20 ppm. A 3′ UMP standard has a single peak at 3.4 ppm. When 2′,3′-cUMP is incubated with AtRNL, which leaves a 2′ phosphate 8 , there is a single peak at 3.2 ppm. Incubation of 2′,3′-cUMP with yUsb1 produces a new signal at 3.4 ppm ( c , bottom ) Zoom of dashed region in top panel. d Time course of Usb1 processing on RNAs with different 3′ end modifications. yUsb1 is most active on RNA substrates with a cis -diol (lanes 1–4), less active on those with a 2′,3′-cyclic phosphate ( > p; lanes 5–8) or 2′ phosphates (2′P; lanes 9–12), and is inactive on 3′ phosphate ends (3′P; lanes 13–16). e Model describing the dual activities of yUsb1
    Figure Legend Snippet: yUsb1 acts as a 3′–5′exonuclease and CPDase in vitro. a U6 snRNA is synthesized by RNA Polymerase III. Transcription termination produces a heterogeneous U6 with a 4–8 nucleotide U-tail. Processing by yUsb1 shortens the U-tail and leaves a phosphoryl group. b Usb1 removes nucleotides from the 3′ end of RNAs. The 5′-labeled U6 95–112+3U oligonucleotide cis -diol substrate (lane 2) is insensitive to CIP (lane 3) or T4 PNK (lane 4) treatment. Incubation with yUsb1 for 1 h results in a shorter product (lane 5). Similar reactivity of the product to both CIP (lane 6) and T4 PNK (lane 7) indicates that the product is a noncyclic phosphate. An alkaline hydrolysis ladder (lane 1) shows the mobility of oligonucleotide products of different lengths. ( c , top ) One-dimensional 31 P NMR spectra of 2′,3′-cUMP shows a single peak at 20 ppm. A 3′ UMP standard has a single peak at 3.4 ppm. When 2′,3′-cUMP is incubated with AtRNL, which leaves a 2′ phosphate 8 , there is a single peak at 3.2 ppm. Incubation of 2′,3′-cUMP with yUsb1 produces a new signal at 3.4 ppm ( c , bottom ) Zoom of dashed region in top panel. d Time course of Usb1 processing on RNAs with different 3′ end modifications. yUsb1 is most active on RNA substrates with a cis -diol (lanes 1–4), less active on those with a 2′,3′-cyclic phosphate ( > p; lanes 5–8) or 2′ phosphates (2′P; lanes 9–12), and is inactive on 3′ phosphate ends (3′P; lanes 13–16). e Model describing the dual activities of yUsb1

    Techniques Used: In Vitro, Synthesized, Labeling, Incubation, Nuclear Magnetic Resonance

    3) Product Images from "Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities"

    Article Title: Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00484-w

    yUsb1 acts as a 3′–5′exonuclease and CPDase in vitro. a U6 snRNA is synthesized by RNA Polymerase III. Transcription termination produces a heterogeneous U6 with a 4–8 nucleotide U-tail. Processing by yUsb1 shortens the U-tail and leaves a phosphoryl group. b Usb1 removes nucleotides from the 3′ end of RNAs. The 5′-labeled U6 95–112+3U oligonucleotide cis -diol substrate (lane 2) is insensitive to CIP (lane 3) or T4 PNK (lane 4) treatment. Incubation with yUsb1 for 1 h results in a shorter product (lane 5). Similar reactivity of the product to both CIP (lane 6) and T4 PNK (lane 7) indicates that the product is a noncyclic phosphate. An alkaline hydrolysis ladder (lane 1) shows the mobility of oligonucleotide products of different lengths. ( c , top ) One-dimensional 31 P NMR spectra of 2′,3′-cUMP shows a single peak at 20 ppm. A 3′ UMP standard has a single peak at 3.4 ppm. When 2′,3′-cUMP is incubated with AtRNL, which leaves a 2′ phosphate 8 , there is a single peak at 3.2 ppm. Incubation of 2′,3′-cUMP with yUsb1 produces a new signal at 3.4 ppm ( c , bottom ) Zoom of dashed region in top panel. d Time course of Usb1 processing on RNAs with different 3′ end modifications. yUsb1 is most active on RNA substrates with a cis -diol (lanes 1–4), less active on those with a 2′,3′-cyclic phosphate ( > p; lanes 5–8) or 2′ phosphates (2′P; lanes 9–12), and is inactive on 3′ phosphate ends (3′P; lanes 13–16). e Model describing the dual activities of yUsb1
    Figure Legend Snippet: yUsb1 acts as a 3′–5′exonuclease and CPDase in vitro. a U6 snRNA is synthesized by RNA Polymerase III. Transcription termination produces a heterogeneous U6 with a 4–8 nucleotide U-tail. Processing by yUsb1 shortens the U-tail and leaves a phosphoryl group. b Usb1 removes nucleotides from the 3′ end of RNAs. The 5′-labeled U6 95–112+3U oligonucleotide cis -diol substrate (lane 2) is insensitive to CIP (lane 3) or T4 PNK (lane 4) treatment. Incubation with yUsb1 for 1 h results in a shorter product (lane 5). Similar reactivity of the product to both CIP (lane 6) and T4 PNK (lane 7) indicates that the product is a noncyclic phosphate. An alkaline hydrolysis ladder (lane 1) shows the mobility of oligonucleotide products of different lengths. ( c , top ) One-dimensional 31 P NMR spectra of 2′,3′-cUMP shows a single peak at 20 ppm. A 3′ UMP standard has a single peak at 3.4 ppm. When 2′,3′-cUMP is incubated with AtRNL, which leaves a 2′ phosphate 8 , there is a single peak at 3.2 ppm. Incubation of 2′,3′-cUMP with yUsb1 produces a new signal at 3.4 ppm ( c , bottom ) Zoom of dashed region in top panel. d Time course of Usb1 processing on RNAs with different 3′ end modifications. yUsb1 is most active on RNA substrates with a cis -diol (lanes 1–4), less active on those with a 2′,3′-cyclic phosphate ( > p; lanes 5–8) or 2′ phosphates (2′P; lanes 9–12), and is inactive on 3′ phosphate ends (3′P; lanes 13–16). e Model describing the dual activities of yUsb1

    Techniques Used: In Vitro, Synthesized, Labeling, Incubation, Nuclear Magnetic Resonance

    4) Product Images from "Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile"

    Article Title: Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt1009

    Lysidine is absent in tRNA 2 Ile from Bacillus subtilis JJS80 lacking tilS . ( A ) 1D TLC analysis of the wobble position 34 in tRNA 2 Ile purified from B. subtilis wild-type and JJS80. Purified wild-type tRNA 2 and mutant tRNA 2 were partially hydrolyzed by alkali, the 5′ termini of the fragments were 32 P-labeled using T4-PNK. 32 P-labeled fragments were subsequently digested with nuclease P1 and the nature of the 5′ terminal nucleotide was determined by TLC. The solvent used was isobutyric acid:concentrated ammonia:water (66:1:33) ( v : v : v ). The mobility of each nucleotide (pA, pC, pG, pU) was confirmed with non-radiolabeled standards used as internal markers and visualized by UV shadowing. ( B and C ) Template-dependent binding of purified wild-type 3 H-Ile-tRNA 2 (B) and mutant 35 S-Met-tRNA 2 (C) to ribosomes isolated from B. subtilis . Oligonucleotides used were AUG AUA, AUG AUC, AUG AUG, AUG AUU and AUG UUU; the oligonucleotide concentration was 200 μM.
    Figure Legend Snippet: Lysidine is absent in tRNA 2 Ile from Bacillus subtilis JJS80 lacking tilS . ( A ) 1D TLC analysis of the wobble position 34 in tRNA 2 Ile purified from B. subtilis wild-type and JJS80. Purified wild-type tRNA 2 and mutant tRNA 2 were partially hydrolyzed by alkali, the 5′ termini of the fragments were 32 P-labeled using T4-PNK. 32 P-labeled fragments were subsequently digested with nuclease P1 and the nature of the 5′ terminal nucleotide was determined by TLC. The solvent used was isobutyric acid:concentrated ammonia:water (66:1:33) ( v : v : v ). The mobility of each nucleotide (pA, pC, pG, pU) was confirmed with non-radiolabeled standards used as internal markers and visualized by UV shadowing. ( B and C ) Template-dependent binding of purified wild-type 3 H-Ile-tRNA 2 (B) and mutant 35 S-Met-tRNA 2 (C) to ribosomes isolated from B. subtilis . Oligonucleotides used were AUG AUA, AUG AUC, AUG AUG, AUG AUU and AUG UUU; the oligonucleotide concentration was 200 μM.

    Techniques Used: Thin Layer Chromatography, Purification, Mutagenesis, Labeling, Binding Assay, Isolation, Concentration Assay

    5) 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

    6) 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

    7) Product Images from "Purification of cross-linked RNA-protein complexes by phenol-toluol extraction"

    Article Title: Purification of cross-linked RNA-protein complexes by phenol-toluol extraction

    Journal: Nature Communications

    doi: 10.1038/s41467-019-08942-3

    PTex recovers bacterial RNPs.  a Salmonella  Typhimurium SL1344 Hfq-FLAG was UV-cross-linked and HOT-PTex was performed to purify bacterial RNPs.  b  Western blot using an anti-FLAG antibody demonstrates recovery of Hfq monomers linked to RNA. Note that the physiologically active Hfq hexamer partially withstands SDS-PAGE conditions  49  and that this complex is also enriched after PTex.  c  RNPs in  Salmonella  were purified by PTex globally. 172 Proteins enriched after UV-cross-linking (PTex CL) contain ribosomal proteins (transparent red), known RBPs (red) and DNA-binders (orange). Individual enriched proteins not known to associate with RNA before were used for validation (in parentheses).  d  Validation of PTex-enriched RNA-interactors:  Salmonella  strains expressing FLAG-tagged proteins were immunoprecipitated  ±UV irradiation. RNA-association is confirmed by radioactive labelling of RNA 5′ ends by polynucleotide kinase (T4 PNK) using autoradiography; a signal is exclusively detectable after UV-cross-linking and radiolabelling of precipitated RNA. CsrA-FLAG (pos. ctr.), YigA-FLAG (neg. ctr.), AhpC-FLAG, SipA-FLAG and YihI-FLAG are bound to RNA in vivo.  e  GO terms significantly enriched among the RNA-associated proteins;  p -value derived from a one-tail Fisher Exact test  75 . For full gels/blots see Supplementary Figures   25 ,   26
    Figure Legend Snippet: PTex recovers bacterial RNPs. a Salmonella Typhimurium SL1344 Hfq-FLAG was UV-cross-linked and HOT-PTex was performed to purify bacterial RNPs. b Western blot using an anti-FLAG antibody demonstrates recovery of Hfq monomers linked to RNA. Note that the physiologically active Hfq hexamer partially withstands SDS-PAGE conditions 49 and that this complex is also enriched after PTex. c RNPs in Salmonella were purified by PTex globally. 172 Proteins enriched after UV-cross-linking (PTex CL) contain ribosomal proteins (transparent red), known RBPs (red) and DNA-binders (orange). Individual enriched proteins not known to associate with RNA before were used for validation (in parentheses). d Validation of PTex-enriched RNA-interactors: Salmonella strains expressing FLAG-tagged proteins were immunoprecipitated  ±UV irradiation. RNA-association is confirmed by radioactive labelling of RNA 5′ ends by polynucleotide kinase (T4 PNK) using autoradiography; a signal is exclusively detectable after UV-cross-linking and radiolabelling of precipitated RNA. CsrA-FLAG (pos. ctr.), YigA-FLAG (neg. ctr.), AhpC-FLAG, SipA-FLAG and YihI-FLAG are bound to RNA in vivo. e GO terms significantly enriched among the RNA-associated proteins; p -value derived from a one-tail Fisher Exact test 75 . For full gels/blots see Supplementary Figures  25 , 26

    Techniques Used: Western Blot, SDS Page, Purification, Expressing, Immunoprecipitation, Irradiation, Autoradiography, In Vivo, Derivative Assay

    8) Product Images from "PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2BGlu2"

    Article Title: PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2BGlu2

    Journal: Nature Communications

    doi: 10.1038/ncomms12404

    PARP3 monoribosylates H2B in damaged chromatin. ( a , left) 10μg of the chicken chromatin employed in these experiments was fractionated by SDS–PAGE and stained with Coomassie blue. (right) One microgram of soluble MNase-treated chicken chromatin or 50-mer oligonucleotide duplex (200 nM) harbouring a nick with 3′-P/5′-OH termini was mock-treated (0) or treated with 1, 0.5 or 0.25 U T4 PNK to restore 3′-OH/5′-P termini. These DNA substrates were then incubated with 100 nM hPARP3 and 12.5 μM biotin-NAD + for 30 min and biotinylated products separated by 15% SDS–PAGE and detected with streptavidin-HRP. ( b ) 1 μg chicken chromatin or the indicated recombinant histone was incubated with 100 nM hPARP3 in the presence of 300 nM 32 P-NAD + or 12.5 μM biotin-NAD and oligonucleotide harbouring either a DSB (middle) or SSB (right) and the reaction products fractionated by 15% SDS–PAGE and detected by autoradiography or streptavidin-HRP. (left) An aliquot of the chicken chromatin and recombinant histones was fractionated by SDS–PAGE and stained with Coomassie blue. ( c , left) Aliquots of recombinant histone standards were fractionated separately or together as an octamer on triton-acid urea gels and analysed by staining with Coomassie blue. (right) The products of the PARP3 ribosylation reactions conducted in b were fractionated on triton-acid urea gels and analysed by autoradiography. HRP, horseradish peroxidase.
    Figure Legend Snippet: PARP3 monoribosylates H2B in damaged chromatin. ( a , left) 10μg of the chicken chromatin employed in these experiments was fractionated by SDS–PAGE and stained with Coomassie blue. (right) One microgram of soluble MNase-treated chicken chromatin or 50-mer oligonucleotide duplex (200 nM) harbouring a nick with 3′-P/5′-OH termini was mock-treated (0) or treated with 1, 0.5 or 0.25 U T4 PNK to restore 3′-OH/5′-P termini. These DNA substrates were then incubated with 100 nM hPARP3 and 12.5 μM biotin-NAD + for 30 min and biotinylated products separated by 15% SDS–PAGE and detected with streptavidin-HRP. ( b ) 1 μg chicken chromatin or the indicated recombinant histone was incubated with 100 nM hPARP3 in the presence of 300 nM 32 P-NAD + or 12.5 μM biotin-NAD and oligonucleotide harbouring either a DSB (middle) or SSB (right) and the reaction products fractionated by 15% SDS–PAGE and detected by autoradiography or streptavidin-HRP. (left) An aliquot of the chicken chromatin and recombinant histones was fractionated by SDS–PAGE and stained with Coomassie blue. ( c , left) Aliquots of recombinant histone standards were fractionated separately or together as an octamer on triton-acid urea gels and analysed by staining with Coomassie blue. (right) The products of the PARP3 ribosylation reactions conducted in b were fractionated on triton-acid urea gels and analysed by autoradiography. HRP, horseradish peroxidase.

    Techniques Used: SDS Page, Staining, Incubation, TNKS1 Histone Ribosylation Assay, Recombinant, Autoradiography

    9) Product Images from "The RNA Binding Specificity of Human APOBEC3 Proteins Resembles That of HIV-1 Nucleocapsid"

    Article Title: The RNA Binding Specificity of Human APOBEC3 Proteins Resembles That of HIV-1 Nucleocapsid

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005833

    Flow diagram of CLIP-seq. Cells are fed with the ribonucleoside analog 4-thiouridine (4SU) which is incorporated into nascent RNA. Live cells are then irradiated with ultraviolet (UV) light, which induces covalent cross-links between proteins and RNA at sites of contact and 4SU incorporation. Cells are then lysed and treated with RNase A to generate oligonucleotides crosslinked to proteins of interest. Protein-RNA complexes are then immunopurified, and then the RNA is dephosphorylated at the 3'-end with alkaline phosphatase. The RNA is subsequently radiolabeled with  32 P using T4 polynucleotide kinase (PNK) for detection by autoradiography. Protein-RNA complexes are then separated by SDS-PAGE, transferred to nitrocellulose and a region corresponding to protein-RNA adducts is excised. Cross-linked RNA is isolated by proteinase K treatment and phenol:chloroform extraction. After sequential adapter ligations, the RNA library is reverse transcribed. Reverse transcriptase often misincorporates a G opposite the 4SU cross-linking site, which leads to T to C substitutions in positive-strand of the cDNA, enabling the precise mapping of protein-RNA interaction sites. After PCR amplification, the cDNA library is sequenced by Illumina sequencing.
    Figure Legend Snippet: Flow diagram of CLIP-seq. Cells are fed with the ribonucleoside analog 4-thiouridine (4SU) which is incorporated into nascent RNA. Live cells are then irradiated with ultraviolet (UV) light, which induces covalent cross-links between proteins and RNA at sites of contact and 4SU incorporation. Cells are then lysed and treated with RNase A to generate oligonucleotides crosslinked to proteins of interest. Protein-RNA complexes are then immunopurified, and then the RNA is dephosphorylated at the 3'-end with alkaline phosphatase. The RNA is subsequently radiolabeled with 32 P using T4 polynucleotide kinase (PNK) for detection by autoradiography. Protein-RNA complexes are then separated by SDS-PAGE, transferred to nitrocellulose and a region corresponding to protein-RNA adducts is excised. Cross-linked RNA is isolated by proteinase K treatment and phenol:chloroform extraction. After sequential adapter ligations, the RNA library is reverse transcribed. Reverse transcriptase often misincorporates a G opposite the 4SU cross-linking site, which leads to T to C substitutions in positive-strand of the cDNA, enabling the precise mapping of protein-RNA interaction sites. After PCR amplification, the cDNA library is sequenced by Illumina sequencing.

    Techniques Used: Flow Cytometry, Cross-linking Immunoprecipitation, Irradiation, Autoradiography, SDS Page, Isolation, Polymerase Chain Reaction, Amplification, cDNA Library Assay, Sequencing

    10) Product Images from "Genome-wide identification of short 2′,3′-cyclic phosphate-containing RNAs and their regulation in aging"

    Article Title: Genome-wide identification of short 2′,3′-cyclic phosphate-containing RNAs and their regulation in aging

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1008469

    Sequencing of cP-RNAs in mouse tissues. (A)  Total RNAs extracted from mouse tissues were subjected to northern blots for the 5′-halves of cyto tRNA LysCUU  and tRNA AspGUC .  (B)  Terminal structures of the 5′-tRNA half were analyzed enzymatically. Total RNA from the mouse lung was treated with CIP, T4 PNK, or acid followed by CIP treatment (HCl + CIP). NT designates the non-treated sample used as a negative control. The treated total RNA was subjected to northern blots targeting the 5′-tRNA AspGUC  half and microRNA-16 (miR-16). miR-16 was investigated as a control RNA containing 5′-P and 3′-OH ends.  (C)  Gel-purified 20–45-nt RNAs were subjected to cP-RNA-seq, which amplified 140–160-bp cDNA products (5′-adapter, 55 bp; 3′-adapter, 63 bp; and thereby estimated inserted sequences, 22–42 bp). The cDNAs in the region designated with a red line were purified and subjected to Illumina sequencing.  (D)  Proportion of cP-RNAs annotated to the indicated RNAs.
    Figure Legend Snippet: Sequencing of cP-RNAs in mouse tissues. (A) Total RNAs extracted from mouse tissues were subjected to northern blots for the 5′-halves of cyto tRNA LysCUU and tRNA AspGUC . (B) Terminal structures of the 5′-tRNA half were analyzed enzymatically. Total RNA from the mouse lung was treated with CIP, T4 PNK, or acid followed by CIP treatment (HCl + CIP). NT designates the non-treated sample used as a negative control. The treated total RNA was subjected to northern blots targeting the 5′-tRNA AspGUC half and microRNA-16 (miR-16). miR-16 was investigated as a control RNA containing 5′-P and 3′-OH ends. (C) Gel-purified 20–45-nt RNAs were subjected to cP-RNA-seq, which amplified 140–160-bp cDNA products (5′-adapter, 55 bp; 3′-adapter, 63 bp; and thereby estimated inserted sequences, 22–42 bp). The cDNAs in the region designated with a red line were purified and subjected to Illumina sequencing. (D) Proportion of cP-RNAs annotated to the indicated RNAs.

    Techniques Used: Sequencing, Northern Blot, Negative Control, Purification, RNA Sequencing Assay, Amplification

    TaqMan RT-qPCR quantification of cP-RNAs. (A) The alignment patterns of cP-RNAs for Ncbp3 mRNA and 28S rRNA. The positions of representative cP-RNAs, cPR-Ncbp3 and cPR-28S, are indicated. (B) The regions from which 5′-tRNA GlyGCC half (5′-GlyGCC), cPR-Ncbp3, and cPR-28S were derived are shown in red in the secondary structure of respective substrate RNAs. Secondary structure of Ncbp3 mRNA was predicted by ViennaRNA Package 2.0 [ 34 ]. (C) The total RNA from 24-week old mouse skeletal muscle, treated with CIP, wild-type (WT) T4 PNK, or mutant (Mut) T4 PNK lacking 3′-dephosphorylation activity, was subjected to TaqMan RT-qPCR. NT designates a non-treated sample used as a negative control. The amounts from WT T4 PNK-treated RNA were set as 1, and relative amounts are indicated. Averages of three experiments with SD values are shown. (D) The expression of cP-RNAs in the lung and skeletal muscle of 24-week old mice were quantified using TaqMan RT-qPCR. The cP-RNA amounts were estimated based on the standard curves shown in S9 Fig . Averages of three independent experiments with SD values are shown.
    Figure Legend Snippet: TaqMan RT-qPCR quantification of cP-RNAs. (A) The alignment patterns of cP-RNAs for Ncbp3 mRNA and 28S rRNA. The positions of representative cP-RNAs, cPR-Ncbp3 and cPR-28S, are indicated. (B) The regions from which 5′-tRNA GlyGCC half (5′-GlyGCC), cPR-Ncbp3, and cPR-28S were derived are shown in red in the secondary structure of respective substrate RNAs. Secondary structure of Ncbp3 mRNA was predicted by ViennaRNA Package 2.0 [ 34 ]. (C) The total RNA from 24-week old mouse skeletal muscle, treated with CIP, wild-type (WT) T4 PNK, or mutant (Mut) T4 PNK lacking 3′-dephosphorylation activity, was subjected to TaqMan RT-qPCR. NT designates a non-treated sample used as a negative control. The amounts from WT T4 PNK-treated RNA were set as 1, and relative amounts are indicated. Averages of three experiments with SD values are shown. (D) The expression of cP-RNAs in the lung and skeletal muscle of 24-week old mice were quantified using TaqMan RT-qPCR. The cP-RNA amounts were estimated based on the standard curves shown in S9 Fig . Averages of three independent experiments with SD values are shown.

    Techniques Used: Quantitative RT-PCR, Derivative Assay, Mutagenesis, De-Phosphorylation Assay, Activity Assay, Negative Control, Expressing, Mouse Assay

    11) Product Images from "Ligation of 2′, 3′‐cyclic phosphate RNAs for the identification of microRNA binding sites"

    Article Title: Ligation of 2′, 3′‐cyclic phosphate RNAs for the identification of microRNA binding sites

    Journal: Febs Letters

    doi: 10.1002/1873-3468.13976

    Ligation of 2′, 3′‐cyclic phosphate miR‐34a‐5p strands to 5′‐OH mRNA mimics in HeLa lysate. (A) Scheme showing principle and different formats of RNA > p ligation. Left: miRNA > p probe bound to a target mRNA inside the RNA‐induced silencing complex (RISC). Right: Alignments of radiolabeled miR‐34a‐5p to LMTK3‐mRNA mimics. (B) Ligation of miR‐34a‐5p > p (22 nt) to different LMTK3 mRNA mimics (64–72 nt) in HeLa lysate. Mir‐34a‐5p > p ligation is shown in lanes 2, 4, 6, 8, and 10. Controls are shown in lanes 11–16. Mir‐34a‐5p > p (2′,3′ cycP +) and miR‐34a‐5p‐3′‐OH (2′,3′ cycP ‐) were 5′‐ 32 P labeled using T4 PNK (3′‐phosphatase minus). Mir‐34a‐5p > p was annealed to different RNA counter‐strands and mixed (8 n m final) with HeLa extract (protein conc. = 2.1 mg·mL −1 ; 0.84 mg·mL −1 final) and a ligation buffer containing KCl (40 n m final), EDTA (pH = 8, 100 µ m final), MgCl 2 (1.2 m m final), DTT (5 m m final), ATP (3 m m final), GTP (0.2 m m final), and RNasin (1 U·µL −1 ; Promega) and incubated at 37 °C for 30 min. In one setup, a bridging ORN b (30 n m , 12 nt) complementarity to each, mir‐34a‐5p > p and LMTK‐3 mimic 8‐8 , was added prior to annealing. Reference oligonucleotides for the respective ligation products (Controls) were chemically synthesized; differences in band intensity may be caused by differences in labeling efficiency. Representative gel shown, n = 3. Full gel shown in Fig. S5 .
    Figure Legend Snippet: Ligation of 2′, 3′‐cyclic phosphate miR‐34a‐5p strands to 5′‐OH mRNA mimics in HeLa lysate. (A) Scheme showing principle and different formats of RNA > p ligation. Left: miRNA > p probe bound to a target mRNA inside the RNA‐induced silencing complex (RISC). Right: Alignments of radiolabeled miR‐34a‐5p to LMTK3‐mRNA mimics. (B) Ligation of miR‐34a‐5p > p (22 nt) to different LMTK3 mRNA mimics (64–72 nt) in HeLa lysate. Mir‐34a‐5p > p ligation is shown in lanes 2, 4, 6, 8, and 10. Controls are shown in lanes 11–16. Mir‐34a‐5p > p (2′,3′ cycP +) and miR‐34a‐5p‐3′‐OH (2′,3′ cycP ‐) were 5′‐ 32 P labeled using T4 PNK (3′‐phosphatase minus). Mir‐34a‐5p > p was annealed to different RNA counter‐strands and mixed (8 n m final) with HeLa extract (protein conc. = 2.1 mg·mL −1 ; 0.84 mg·mL −1 final) and a ligation buffer containing KCl (40 n m final), EDTA (pH = 8, 100 µ m final), MgCl 2 (1.2 m m final), DTT (5 m m final), ATP (3 m m final), GTP (0.2 m m final), and RNasin (1 U·µL −1 ; Promega) and incubated at 37 °C for 30 min. In one setup, a bridging ORN b (30 n m , 12 nt) complementarity to each, mir‐34a‐5p > p and LMTK‐3 mimic 8‐8 , was added prior to annealing. Reference oligonucleotides for the respective ligation products (Controls) were chemically synthesized; differences in band intensity may be caused by differences in labeling efficiency. Representative gel shown, n = 3. Full gel shown in Fig. S5 .

    Techniques Used: Ligation, Labeling, Incubation, Synthesized

    12) Product Images from "Circadian and feeding rhythms differentially affect rhythmic mRNA transcription and translation in mouse liver"

    Article Title: Circadian and feeding rhythms differentially affect rhythmic mRNA transcription and translation in mouse liver

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

    doi: 10.1073/pnas.1515308112

    Technical validation of ribosome profiling experiments. ( A ) Simplified representation of the modified ribosome profiling method. RNase I digestion leaves a 5′-OH and a 3′-cyclophosphate. T4 polynucleotide kinase treatment performed in
    Figure Legend Snippet: Technical validation of ribosome profiling experiments. ( A ) Simplified representation of the modified ribosome profiling method. RNase I digestion leaves a 5′-OH and a 3′-cyclophosphate. T4 polynucleotide kinase treatment performed in

    Techniques Used: Modification

    13) 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

    14) Product Images from "High-throughput determination of RNA structure by proximity ligation"

    Article Title: High-throughput determination of RNA structure by proximity ligation

    Journal: Nature biotechnology

    doi: 10.1038/nbt.3289

    RNA Proximity Ligation identifies structurally proximate regions within the complex secondary structures of S. cerevisiae ribosomal RNAs. a.) A schematic representation of the RPL method. Whole cells are spheroplasted with zymolyase and RNA is allowed to react with endogenous RNases. RNA ends are repaired in situ via T4 PNK to yield 5′-phosphate termini. Complexes are ligated overnight in the presence of T4 RNA Ligase I. Ligation products are cleaned up via acid guanidinium-phenol and subsequent DNase treatment, and subjected to Illumina TruSeq RNA-seq library preparation. These libraries are sequenced to map and count ligation junctions; b.-c.) We examined the distribution of ligation junctions as a function of distance from known base-pair partners in the 25S/5.8S rRNA and 18S rRNAs. Ligation products capture the structural proximity implied by base-pairing relationships, as evidenced by the enrichment for ligation junctions immediately near paired bases. Y-axes are shown as ligation counts per million reads analyzed. d.) Contact probability map for the eukaryotic 5.8S/25S rRNA based on RPL scores, which are calculated from the frequencies of ligation events between pairs of 21 nt windows ( Methods ). Lower inset : Ligation events, shown for bases 1300 to 1475 of the LSU rRNA in orange, primarily occur across digested single-stranded loops. RPL scores effectively smooth this noisy signal and are enriched for pairs of interacting regions. Plotted here are the 8,463 ligation events where both nucleotides fall within the displayed domain (compared to 17,029 ligation events where one nucleotide falls within the displayed domain and one does not, not shown). Right inset: RPL scores localize known pseudo-knots in the LSU rRNA structure, such as the interaction between bases 1727-1812 (shown in red) and bases 1941 – 2038 (shown in blue).
    Figure Legend Snippet: RNA Proximity Ligation identifies structurally proximate regions within the complex secondary structures of S. cerevisiae ribosomal RNAs. a.) A schematic representation of the RPL method. Whole cells are spheroplasted with zymolyase and RNA is allowed to react with endogenous RNases. RNA ends are repaired in situ via T4 PNK to yield 5′-phosphate termini. Complexes are ligated overnight in the presence of T4 RNA Ligase I. Ligation products are cleaned up via acid guanidinium-phenol and subsequent DNase treatment, and subjected to Illumina TruSeq RNA-seq library preparation. These libraries are sequenced to map and count ligation junctions; b.-c.) We examined the distribution of ligation junctions as a function of distance from known base-pair partners in the 25S/5.8S rRNA and 18S rRNAs. Ligation products capture the structural proximity implied by base-pairing relationships, as evidenced by the enrichment for ligation junctions immediately near paired bases. Y-axes are shown as ligation counts per million reads analyzed. d.) Contact probability map for the eukaryotic 5.8S/25S rRNA based on RPL scores, which are calculated from the frequencies of ligation events between pairs of 21 nt windows ( Methods ). Lower inset : Ligation events, shown for bases 1300 to 1475 of the LSU rRNA in orange, primarily occur across digested single-stranded loops. RPL scores effectively smooth this noisy signal and are enriched for pairs of interacting regions. Plotted here are the 8,463 ligation events where both nucleotides fall within the displayed domain (compared to 17,029 ligation events where one nucleotide falls within the displayed domain and one does not, not shown). Right inset: RPL scores localize known pseudo-knots in the LSU rRNA structure, such as the interaction between bases 1727-1812 (shown in red) and bases 1941 – 2038 (shown in blue).

    Techniques Used: Ligation, In Situ, RNA Sequencing Assay

    15) Product Images from "The wobble nucleotide-excising anticodon nuclease RloC is governed by the zinc-hook and DNA-dependent ATPase of its Rad50-like region"

    Article Title: The wobble nucleotide-excising anticodon nuclease RloC is governed by the zinc-hook and DNA-dependent ATPase of its Rad50-like region

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks593

    Gka RloC’s ATPase activates its ACNase. Gka RloC’s ACNase of the IMAC fraction was assayed in vitro in panels ( A )–( C ) and ( E ) essentially as described in Materials and Methods but in the absence of added DNA. ( A ) Dependence of Gka RloC’s ACNase activity on ATP’s level. ( B ) Gka RloC’s ACNase activity was assayed in the presence of 500 µM of the indicated nucleotides. ( C ) Time courses of Gka RloC’s ACNase activity in the presence of 0.5 mM ATP and indicated amounts of AMPPNP. ( D ) In vivo ACNase activity of the indicated Gka RloC alleles. Left panel—RNA extracted from cells expressing these alleles was 5′-end labelled using T4 Pnk and separated by denaturing PAGE. Right panel—the expression of the indicated Gka RloC alleles were monitored by Western using an anti-His tag monoclonal antibody ( 4 ). ( E ) Nucleotide specificity of Gka RloC’s ACNase activation. The activation reaction was performed in the presence of the indicated nucleotides (GTP and ATP at 0.5 mM each, dTTP at 5 µM).
    Figure Legend Snippet: Gka RloC’s ATPase activates its ACNase. Gka RloC’s ACNase of the IMAC fraction was assayed in vitro in panels ( A )–( C ) and ( E ) essentially as described in Materials and Methods but in the absence of added DNA. ( A ) Dependence of Gka RloC’s ACNase activity on ATP’s level. ( B ) Gka RloC’s ACNase activity was assayed in the presence of 500 µM of the indicated nucleotides. ( C ) Time courses of Gka RloC’s ACNase activity in the presence of 0.5 mM ATP and indicated amounts of AMPPNP. ( D ) In vivo ACNase activity of the indicated Gka RloC alleles. Left panel—RNA extracted from cells expressing these alleles was 5′-end labelled using T4 Pnk and separated by denaturing PAGE. Right panel—the expression of the indicated Gka RloC alleles were monitored by Western using an anti-His tag monoclonal antibody ( 4 ). ( E ) Nucleotide specificity of Gka RloC’s ACNase activation. The activation reaction was performed in the presence of the indicated nucleotides (GTP and ATP at 0.5 mM each, dTTP at 5 µM).

    Techniques Used: In Vitro, Activity Assay, In Vivo, Expressing, Polyacrylamide Gel Electrophoresis, Western Blot, Activation Assay

    16) Product Images from "Tagsteady: a metabarcoding library preparation protocol to avoid false assignment of sequences to samples"

    Article Title: Tagsteady: a metabarcoding library preparation protocol to avoid false assignment of sequences to samples

    Journal: bioRxiv

    doi: 10.1101/2020.01.22.915009

    Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, T4 PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).
    Figure Legend Snippet: Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, T4 PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).

    Techniques Used: Polymerase Chain Reaction, Activity Assay, Sequencing, Ligation, Amplification

    17) Product Images from "Capped small RNAs and MOV10 in Human Hepatitis Delta Virus replication"

    Article Title: Capped small RNAs and MOV10 in Human Hepatitis Delta Virus replication

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.1440

    The antigenomic HDV small RNA is 2′-3′ hydroxylated and has an mRNA-like 5′ cap (Northern Blot, 293 cells, RNA induction). ( a ) 3′ end by β-elimination. The mobility of the HDV small RNA is increased following β-elimination. miR-15a: 2′-3′ hydroxylated positive control; +β: +β-elimination; -β: untreated RNA. ( b ) 5′ end by enzymatic analysis. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Decapping enzyme (TAP; +HDV); 5: T4 RNA Ligase (+HDV); 6: Terminator Exonuclease (+HDV). The size of the HDV small RNA was estimated to be ∼24nt based on the largely 22nt, 5′ phosphorylated miR15-a shown in the inset (IS). ( c ) Confirmation that the 5′ end of the HDV small RNA is capped, not triphosphorylated (enlarged image to emphasize changes in gel mobility for miR-15a, but not HDV small RNA). 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: Antarctic Phosphatase (+HDV); 4: Antarctic Phosphatase followed by T4 PNK (+HDV). ( d ) RNA immunoprecipitation with anti-2,2,7-trimethylguanosine antibody K121. The immunoprecipitation efficiency of the HDV small RNA, U5 snRNA (positive control) and microRNAs miR-15a and let-7a (negative controls) was analysed by Northern blot. ‘S’: supernatant; ‘I’: IP fraction. ( e ) Predicted structure of the HDV small RNA. The various RNAs in a - d were detected after stripping and rehybridisation to the same blot. M: RNA marker.
    Figure Legend Snippet: The antigenomic HDV small RNA is 2′-3′ hydroxylated and has an mRNA-like 5′ cap (Northern Blot, 293 cells, RNA induction). ( a ) 3′ end by β-elimination. The mobility of the HDV small RNA is increased following β-elimination. miR-15a: 2′-3′ hydroxylated positive control; +β: +β-elimination; -β: untreated RNA. ( b ) 5′ end by enzymatic analysis. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Decapping enzyme (TAP; +HDV); 5: T4 RNA Ligase (+HDV); 6: Terminator Exonuclease (+HDV). The size of the HDV small RNA was estimated to be ∼24nt based on the largely 22nt, 5′ phosphorylated miR15-a shown in the inset (IS). ( c ) Confirmation that the 5′ end of the HDV small RNA is capped, not triphosphorylated (enlarged image to emphasize changes in gel mobility for miR-15a, but not HDV small RNA). 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: Antarctic Phosphatase (+HDV); 4: Antarctic Phosphatase followed by T4 PNK (+HDV). ( d ) RNA immunoprecipitation with anti-2,2,7-trimethylguanosine antibody K121. The immunoprecipitation efficiency of the HDV small RNA, U5 snRNA (positive control) and microRNAs miR-15a and let-7a (negative controls) was analysed by Northern blot. ‘S’: supernatant; ‘I’: IP fraction. ( e ) Predicted structure of the HDV small RNA. The various RNAs in a - d were detected after stripping and rehybridisation to the same blot. M: RNA marker.

    Techniques Used: Northern Blot, Positive Control, Immunoprecipitation, Stripping Membranes, Marker

    Cloning and characterization of an HDV small RNA of genomic polarity. ( a ) Relative location and cloning frequency of sequenced HDV small RNAs derived from the genomic and antigenomic pode hairpins (main species highlighted in red). ( b ) Detection of genomic HDV small RNA by Northern Blot (293 cells, day 5). 1: DNA induction, wt HDAg; 2: DNA induction, mutant HDAg. ( c ) Enzymatic analysis of genomic small RNA 5′ end. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Antarctic Phosphatase (+HDV); 5: Antarctic Phosphatase followed by T4 PNK (+HDV); 6: Decapping enzyme (TAP; +HDV); 7: T4 RNA Ligase (+HDV); 8: Terminator Exonuclease (+HDV). Note that unlike the antigenomic small RNA, a minor fraction of the genomic small RNA does not appear to be shifted following TAP treatment. ( d - f ) Localization of the HDV small RNAs. ( d ) Nuclear-cytoplasmic fractionation of antigenomic HDV small RNA (polyacrylamide gel) and full-length antigenomic and genomic HDV RNA (denaturing agarose gel). The main species in the full-length genomic/antigenomic RNA blot corresponds to the monomer, the higher molecular weight species to dimer, trimer etc. 1: DNA induction, mutant HDAg; 2: DNA induction, wt HDAg; 3: untransfected. ( e ) Genomic small RNA is restricted to the nucleus (nuclear-cytoplasmic fractionation). 1: DNA induction, wt HDAg; 2: DNA induction, mutant HDAg. miR-15a and U6 snRNA chosen as largely cytoplasmic and nuclear RNA controls, respectively. ( f ) The HDV small RNA can be found in the HDV virion. 1: RNA induction (same RNA as in Fig. 2c ); 2: virion RNA isolated from tissue culture media (∼1.25×10 9 particles). MR: RNA Marker. The various RNAs in c-f were detected after stripping and re-hybridization to the same blot.
    Figure Legend Snippet: Cloning and characterization of an HDV small RNA of genomic polarity. ( a ) Relative location and cloning frequency of sequenced HDV small RNAs derived from the genomic and antigenomic pode hairpins (main species highlighted in red). ( b ) Detection of genomic HDV small RNA by Northern Blot (293 cells, day 5). 1: DNA induction, wt HDAg; 2: DNA induction, mutant HDAg. ( c ) Enzymatic analysis of genomic small RNA 5′ end. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Antarctic Phosphatase (+HDV); 5: Antarctic Phosphatase followed by T4 PNK (+HDV); 6: Decapping enzyme (TAP; +HDV); 7: T4 RNA Ligase (+HDV); 8: Terminator Exonuclease (+HDV). Note that unlike the antigenomic small RNA, a minor fraction of the genomic small RNA does not appear to be shifted following TAP treatment. ( d - f ) Localization of the HDV small RNAs. ( d ) Nuclear-cytoplasmic fractionation of antigenomic HDV small RNA (polyacrylamide gel) and full-length antigenomic and genomic HDV RNA (denaturing agarose gel). The main species in the full-length genomic/antigenomic RNA blot corresponds to the monomer, the higher molecular weight species to dimer, trimer etc. 1: DNA induction, mutant HDAg; 2: DNA induction, wt HDAg; 3: untransfected. ( e ) Genomic small RNA is restricted to the nucleus (nuclear-cytoplasmic fractionation). 1: DNA induction, wt HDAg; 2: DNA induction, mutant HDAg. miR-15a and U6 snRNA chosen as largely cytoplasmic and nuclear RNA controls, respectively. ( f ) The HDV small RNA can be found in the HDV virion. 1: RNA induction (same RNA as in Fig. 2c ); 2: virion RNA isolated from tissue culture media (∼1.25×10 9 particles). MR: RNA Marker. The various RNAs in c-f were detected after stripping and re-hybridization to the same blot.

    Techniques Used: Clone Assay, Derivative Assay, Northern Blot, Mutagenesis, Fractionation, Agarose Gel Electrophoresis, Northern blot, Molecular Weight, Isolation, Marker, Stripping Membranes, Hybridization

    18) Product Images from "Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities"

    Article Title: Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00484-w

    yUsb1 acts as a 3′–5′exonuclease and CPDase in vitro. a U6 snRNA is synthesized by RNA Polymerase III. Transcription termination produces a heterogeneous U6 with a 4–8 nucleotide U-tail. Processing by yUsb1 shortens the U-tail and leaves a phosphoryl group. b Usb1 removes nucleotides from the 3′ end of RNAs. The 5′-labeled U6 95–112+3U oligonucleotide cis -diol substrate (lane 2) is insensitive to CIP (lane 3) or T4 PNK (lane 4) treatment. Incubation with yUsb1 for 1 h results in a shorter product (lane 5). Similar reactivity of the product to both CIP (lane 6) and T4 PNK (lane 7) indicates that the product is a noncyclic phosphate. An alkaline hydrolysis ladder (lane 1) shows the mobility of oligonucleotide products of different lengths. ( c , top ) One-dimensional 31 P NMR spectra of 2′,3′-cUMP shows a single peak at 20 ppm. A 3′ UMP standard has a single peak at 3.4 ppm. When 2′,3′-cUMP is incubated with AtRNL, which leaves a 2′ phosphate 8 , there is a single peak at 3.2 ppm. Incubation of 2′,3′-cUMP with yUsb1 produces a new signal at 3.4 ppm ( c , bottom ) Zoom of dashed region in top panel. d Time course of Usb1 processing on RNAs with different 3′ end modifications. yUsb1 is most active on RNA substrates with a cis -diol (lanes 1–4), less active on those with a 2′,3′-cyclic phosphate ( > p; lanes 5–8) or 2′ phosphates (2′P; lanes 9–12), and is inactive on 3′ phosphate ends (3′P; lanes 13–16). e Model describing the dual activities of yUsb1
    Figure Legend Snippet: yUsb1 acts as a 3′–5′exonuclease and CPDase in vitro. a U6 snRNA is synthesized by RNA Polymerase III. Transcription termination produces a heterogeneous U6 with a 4–8 nucleotide U-tail. Processing by yUsb1 shortens the U-tail and leaves a phosphoryl group. b Usb1 removes nucleotides from the 3′ end of RNAs. The 5′-labeled U6 95–112+3U oligonucleotide cis -diol substrate (lane 2) is insensitive to CIP (lane 3) or T4 PNK (lane 4) treatment. Incubation with yUsb1 for 1 h results in a shorter product (lane 5). Similar reactivity of the product to both CIP (lane 6) and T4 PNK (lane 7) indicates that the product is a noncyclic phosphate. An alkaline hydrolysis ladder (lane 1) shows the mobility of oligonucleotide products of different lengths. ( c , top ) One-dimensional 31 P NMR spectra of 2′,3′-cUMP shows a single peak at 20 ppm. A 3′ UMP standard has a single peak at 3.4 ppm. When 2′,3′-cUMP is incubated with AtRNL, which leaves a 2′ phosphate 8 , there is a single peak at 3.2 ppm. Incubation of 2′,3′-cUMP with yUsb1 produces a new signal at 3.4 ppm ( c , bottom ) Zoom of dashed region in top panel. d Time course of Usb1 processing on RNAs with different 3′ end modifications. yUsb1 is most active on RNA substrates with a cis -diol (lanes 1–4), less active on those with a 2′,3′-cyclic phosphate ( > p; lanes 5–8) or 2′ phosphates (2′P; lanes 9–12), and is inactive on 3′ phosphate ends (3′P; lanes 13–16). e Model describing the dual activities of yUsb1

    Techniques Used: In Vitro, Synthesized, Labeling, Incubation, Nuclear Magnetic Resonance

    19) Product Images from "Circadian and feeding rhythms differentially affect rhythmic mRNA transcription and translation in mouse liver"

    Article Title: Circadian and feeding rhythms differentially affect rhythmic mRNA transcription and translation in mouse liver

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

    doi: 10.1073/pnas.1515308112

    Technical validation of ribosome profiling experiments. ( A ) Simplified representation of the modified ribosome profiling method. RNase I digestion leaves a 5′-OH and a 3′-cyclophosphate. T4 polynucleotide kinase treatment performed in
    Figure Legend Snippet: Technical validation of ribosome profiling experiments. ( A ) Simplified representation of the modified ribosome profiling method. RNase I digestion leaves a 5′-OH and a 3′-cyclophosphate. T4 polynucleotide kinase treatment performed in

    Techniques Used: Modification

    20) Product Images from "Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)"

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

    Journal: RNA

    doi: 10.1261/rna.5247704

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

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

    21) Product Images from "Capped small RNAs and MOV10 in Human Hepatitis Delta Virus replication"

    Article Title: Capped small RNAs and MOV10 in Human Hepatitis Delta Virus replication

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.1440

    The antigenomic HDV small RNA is 2′-3′ hydroxylated and has an mRNA-like 5′ cap (Northern Blot, 293 cells, RNA induction). ( a ) 3′ end by β-elimination. The mobility of the HDV small RNA is increased following β-elimination. miR-15a: 2′-3′ hydroxylated positive control; +β: +β-elimination; -β: untreated RNA. ( b ) 5′ end by enzymatic analysis. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Decapping enzyme (TAP; +HDV); 5: T4 RNA Ligase (+HDV); 6: Terminator Exonuclease (+HDV). The size of the HDV small RNA was estimated to be ∼24nt based on the largely 22nt, 5′ phosphorylated miR15-a shown in the inset (IS). ( c ) Confirmation that the 5′ end of the HDV small RNA is capped, not triphosphorylated (enlarged image to emphasize changes in gel mobility for miR-15a, but not HDV small RNA). 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: Antarctic Phosphatase (+HDV); 4: Antarctic Phosphatase followed by T4 PNK (+HDV). ( d ) RNA immunoprecipitation with anti-2,2,7-trimethylguanosine antibody K121. The immunoprecipitation efficiency of the HDV small RNA, U5 snRNA (positive control) and microRNAs miR-15a and let-7a (negative controls) was analysed by Northern blot. ‘S’: supernatant; ‘I’: IP fraction. ( e ) Predicted structure of the HDV small RNA. The various RNAs in a - d were detected after stripping and rehybridisation to the same blot. M: RNA marker.
    Figure Legend Snippet: The antigenomic HDV small RNA is 2′-3′ hydroxylated and has an mRNA-like 5′ cap (Northern Blot, 293 cells, RNA induction). ( a ) 3′ end by β-elimination. The mobility of the HDV small RNA is increased following β-elimination. miR-15a: 2′-3′ hydroxylated positive control; +β: +β-elimination; -β: untreated RNA. ( b ) 5′ end by enzymatic analysis. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Decapping enzyme (TAP; +HDV); 5: T4 RNA Ligase (+HDV); 6: Terminator Exonuclease (+HDV). The size of the HDV small RNA was estimated to be ∼24nt based on the largely 22nt, 5′ phosphorylated miR15-a shown in the inset (IS). ( c ) Confirmation that the 5′ end of the HDV small RNA is capped, not triphosphorylated (enlarged image to emphasize changes in gel mobility for miR-15a, but not HDV small RNA). 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: Antarctic Phosphatase (+HDV); 4: Antarctic Phosphatase followed by T4 PNK (+HDV). ( d ) RNA immunoprecipitation with anti-2,2,7-trimethylguanosine antibody K121. The immunoprecipitation efficiency of the HDV small RNA, U5 snRNA (positive control) and microRNAs miR-15a and let-7a (negative controls) was analysed by Northern blot. ‘S’: supernatant; ‘I’: IP fraction. ( e ) Predicted structure of the HDV small RNA. The various RNAs in a - d were detected after stripping and rehybridisation to the same blot. M: RNA marker.

    Techniques Used: Northern Blot, Positive Control, Immunoprecipitation, Stripping Membranes, Marker

    Cloning and characterization of an HDV small RNA of genomic polarity. ( a ) Relative location and cloning frequency of sequenced HDV small RNAs derived from the genomic and antigenomic pode hairpins (main species highlighted in red). ( b ) Detection of genomic HDV small RNA by Northern Blot (293 cells, day 5). 1: DNA induction, wt HDAg; 2: DNA induction, mutant HDAg. ( c ) Enzymatic analysis of genomic small RNA 5′ end. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Antarctic Phosphatase (+HDV); 5: Antarctic Phosphatase followed by T4 PNK (+HDV); 6: Decapping enzyme (TAP; +HDV); 7: T4 RNA Ligase (+HDV); 8: Terminator Exonuclease (+HDV). Note that unlike the antigenomic small RNA, a minor fraction of the genomic small RNA does not appear to be shifted following TAP treatment. ( d - f ) Localization of the HDV small RNAs. ( d ) Nuclear-cytoplasmic fractionation of antigenomic HDV small RNA (polyacrylamide gel) and full-length antigenomic and genomic HDV RNA (denaturing agarose gel). The main species in the full-length genomic/antigenomic RNA blot corresponds to the monomer, the higher molecular weight species to dimer, trimer etc. 1: DNA induction, mutant HDAg; 2: DNA induction, wt HDAg; 3: untransfected. ( e ) Genomic small RNA is restricted to the nucleus (nuclear-cytoplasmic fractionation). 1: DNA induction, wt HDAg; 2: DNA induction, mutant HDAg. miR-15a and U6 snRNA chosen as largely cytoplasmic and nuclear RNA controls, respectively. ( f ) The HDV small RNA can be found in the HDV virion. 1: RNA induction (same RNA as in Fig. 2c ); 2: virion RNA isolated from tissue culture media (∼1.25×10 9 particles). MR: RNA Marker. The various RNAs in c-f were detected after stripping and re-hybridization to the same blot.
    Figure Legend Snippet: Cloning and characterization of an HDV small RNA of genomic polarity. ( a ) Relative location and cloning frequency of sequenced HDV small RNAs derived from the genomic and antigenomic pode hairpins (main species highlighted in red). ( b ) Detection of genomic HDV small RNA by Northern Blot (293 cells, day 5). 1: DNA induction, wt HDAg; 2: DNA induction, mutant HDAg. ( c ) Enzymatic analysis of genomic small RNA 5′ end. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Antarctic Phosphatase (+HDV); 5: Antarctic Phosphatase followed by T4 PNK (+HDV); 6: Decapping enzyme (TAP; +HDV); 7: T4 RNA Ligase (+HDV); 8: Terminator Exonuclease (+HDV). Note that unlike the antigenomic small RNA, a minor fraction of the genomic small RNA does not appear to be shifted following TAP treatment. ( d - f ) Localization of the HDV small RNAs. ( d ) Nuclear-cytoplasmic fractionation of antigenomic HDV small RNA (polyacrylamide gel) and full-length antigenomic and genomic HDV RNA (denaturing agarose gel). The main species in the full-length genomic/antigenomic RNA blot corresponds to the monomer, the higher molecular weight species to dimer, trimer etc. 1: DNA induction, mutant HDAg; 2: DNA induction, wt HDAg; 3: untransfected. ( e ) Genomic small RNA is restricted to the nucleus (nuclear-cytoplasmic fractionation). 1: DNA induction, wt HDAg; 2: DNA induction, mutant HDAg. miR-15a and U6 snRNA chosen as largely cytoplasmic and nuclear RNA controls, respectively. ( f ) The HDV small RNA can be found in the HDV virion. 1: RNA induction (same RNA as in Fig. 2c ); 2: virion RNA isolated from tissue culture media (∼1.25×10 9 particles). MR: RNA Marker. The various RNAs in c-f were detected after stripping and re-hybridization to the same blot.

    Techniques Used: Clone Assay, Derivative Assay, Northern Blot, Mutagenesis, Fractionation, Agarose Gel Electrophoresis, Northern blot, Molecular Weight, Isolation, Marker, Stripping Membranes, Hybridization

    22) Product Images from "Evidence that base stacking potential in annealed 3' overhangs determines polymerase utilization in yeast nonhomologous end joining"

    Article Title: Evidence that base stacking potential in annealed 3' overhangs determines polymerase utilization in yeast nonhomologous end joining

    Journal:

    doi: 10.1016/j.dnarep.2007.07.018

    5’ dRP lesions demand gap filling by Pol4, but do not require the Pol4 lyase activity or Rad27. (A) Schematic for construction of OMPs with 5’ dRP termini. Deoxyuracil residues are indicated in gray. Treatment with T4 PNK followed by UDG
    Figure Legend Snippet: 5’ dRP lesions demand gap filling by Pol4, but do not require the Pol4 lyase activity or Rad27. (A) Schematic for construction of OMPs with 5’ dRP termini. Deoxyuracil residues are indicated in gray. Treatment with T4 PNK followed by UDG

    Techniques Used: Activity Assay

    23) Product Images from "Evidence that base stacking potential in annealed 3' overhangs determines polymerase utilization in yeast nonhomologous end joining"

    Article Title: Evidence that base stacking potential in annealed 3' overhangs determines polymerase utilization in yeast nonhomologous end joining

    Journal:

    doi: 10.1016/j.dnarep.2007.07.018

    5’ dRP lesions demand gap filling by Pol4, but do not require the Pol4 lyase activity or Rad27. (A) Schematic for construction of OMPs with 5’ dRP termini. Deoxyuracil residues are indicated in gray. Treatment with T4 PNK followed by UDG
    Figure Legend Snippet: 5’ dRP lesions demand gap filling by Pol4, but do not require the Pol4 lyase activity or Rad27. (A) Schematic for construction of OMPs with 5’ dRP termini. Deoxyuracil residues are indicated in gray. Treatment with T4 PNK followed by UDG

    Techniques Used: Activity Assay

    24) 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

    25) Product Images from "Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts"

    Article Title: Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts

    Journal: Nature biotechnology

    doi: 10.1038/s41587-019-0090-6

    Tornado expression system generates circular RNA a , Ribozymes efficiently self-cleave during transcription reactions.. The construct containing Twister P1 and Twister P3 U2A ribozymes was transcribed  in vitro  and quenched with urea before running on denaturing PAGE and visualizing RNA. Fully cleaved products and the side products of cleavage accumulate efficiently and rapidly after transcription. b , Fully-cleaved products of transcription in  a  contain appropriate ends for circularization by the endogenous ligase, RtcB. We excised the fully-cleaved RNA from  a  and performed an RtcB ligation reactions. RtcB treatment produces a shift in gel mobility that is not observed without ligation or with pre-treatment with T4 PNK. This shift in gel mobility suggests that the fully-cleaved RNA contains the appropriate ends for ligation. Staining of the gel with DFHBI-1T and comparison of fluorescence relative to SYBR Gold signal demonstrates that circular Broccoli is brighter than linear Broccoli. c , Twister-based ribozyme-assisted circular RNA (racRNA) expression generates significantly higher levels of circular RNA than the previous circular RNA expressing system. HEK293T cells expressed racRNA Broccoli from a variety of racRNA expression systems (see  Fig. 1 ) with different combinations of 5’ and 3’ ribozymes and were compared to expression using the tricY system. Cells were treated with actinomycin D (ActD) for 6 h to observe the drop in RNA levels after new RNA synthesis was inhibited. The Twister P1 and Twister P3 U2A construct, dubbed “Tornado”, expresses high levels of Broccoli RNA that exhibit high stability, characteristic of circRNA. d , Tornado-expressed RNA is decisively circular. DNA-directed cleavage by RNase H of a linear RNA produces two bands, each of expected size given the transcript length and probe site. The identical treatment of the same sequence expressed from Tornado produces a single band similar in size to the uncleaved transcribed sample.
    Figure Legend Snippet: Tornado expression system generates circular RNA a , Ribozymes efficiently self-cleave during transcription reactions.. The construct containing Twister P1 and Twister P3 U2A ribozymes was transcribed in vitro and quenched with urea before running on denaturing PAGE and visualizing RNA. Fully cleaved products and the side products of cleavage accumulate efficiently and rapidly after transcription. b , Fully-cleaved products of transcription in a contain appropriate ends for circularization by the endogenous ligase, RtcB. We excised the fully-cleaved RNA from a and performed an RtcB ligation reactions. RtcB treatment produces a shift in gel mobility that is not observed without ligation or with pre-treatment with T4 PNK. This shift in gel mobility suggests that the fully-cleaved RNA contains the appropriate ends for ligation. Staining of the gel with DFHBI-1T and comparison of fluorescence relative to SYBR Gold signal demonstrates that circular Broccoli is brighter than linear Broccoli. c , Twister-based ribozyme-assisted circular RNA (racRNA) expression generates significantly higher levels of circular RNA than the previous circular RNA expressing system. HEK293T cells expressed racRNA Broccoli from a variety of racRNA expression systems (see Fig. 1 ) with different combinations of 5’ and 3’ ribozymes and were compared to expression using the tricY system. Cells were treated with actinomycin D (ActD) for 6 h to observe the drop in RNA levels after new RNA synthesis was inhibited. The Twister P1 and Twister P3 U2A construct, dubbed “Tornado”, expresses high levels of Broccoli RNA that exhibit high stability, characteristic of circRNA. d , Tornado-expressed RNA is decisively circular. DNA-directed cleavage by RNase H of a linear RNA produces two bands, each of expected size given the transcript length and probe site. The identical treatment of the same sequence expressed from Tornado produces a single band similar in size to the uncleaved transcribed sample.

    Techniques Used: Expressing, Construct, In Vitro, Polyacrylamide Gel Electrophoresis, Ligation, Staining, Fluorescence, Sequencing

    26) Product Images from "Perturbation of base excision repair sensitizes breast cancer cells to APOBEC3 deaminase-mediated mutations"

    Article Title: Perturbation of base excision repair sensitizes breast cancer cells to APOBEC3 deaminase-mediated mutations

    Journal: eLife

    doi: 10.7554/eLife.51605

    Purification and activity assays of PNKP and Polβ. ( A ) Purified Polβ-His 6 (17 ng) and PNKP-His 6 (127 ng) from E. coli were subjected to PAGE and stained with Coomassie blue. ( B ) Incorporation of [α- 32 P]-dCTP by Polβ using APE1-generated product. ddC, di-deoxynucleotide; P, product. ( C ) Schematic of the preparation of S (substrate) and subsequent enzymatic reactions for testing PNKP activity. ( D ) Efficiency of oligonucleotide labeling, annealing, and ligation leading to S indicated in ( C ). ( E ) Fpg (NEB, 1 U) completely digested S and the 3’ phosphate was completely removed by PNKP (12.7 ng and 127 ng, lanes 2 and 3), or by T4 PNK (NEB, 0.1 U and 1 U, lanes 7 and 8). NEIL2 (272 ng) only partially digested S and its 3’P was resistant to the PNKP phosphatase (lanes 4 and 5).
    Figure Legend Snippet: Purification and activity assays of PNKP and Polβ. ( A ) Purified Polβ-His 6 (17 ng) and PNKP-His 6 (127 ng) from E. coli were subjected to PAGE and stained with Coomassie blue. ( B ) Incorporation of [α- 32 P]-dCTP by Polβ using APE1-generated product. ddC, di-deoxynucleotide; P, product. ( C ) Schematic of the preparation of S (substrate) and subsequent enzymatic reactions for testing PNKP activity. ( D ) Efficiency of oligonucleotide labeling, annealing, and ligation leading to S indicated in ( C ). ( E ) Fpg (NEB, 1 U) completely digested S and the 3’ phosphate was completely removed by PNKP (12.7 ng and 127 ng, lanes 2 and 3), or by T4 PNK (NEB, 0.1 U and 1 U, lanes 7 and 8). NEIL2 (272 ng) only partially digested S and its 3’P was resistant to the PNKP phosphatase (lanes 4 and 5).

    Techniques Used: Purification, Activity Assay, Polyacrylamide Gel Electrophoresis, Staining, Generated, Oligonucleotide Labeling, Ligation

    27) Product Images from "Ligation of 2′, 3′‐cyclic phosphate RNAs for the identification of microRNA binding sites"

    Article Title: Ligation of 2′, 3′‐cyclic phosphate RNAs for the identification of microRNA binding sites

    Journal: Febs Letters

    doi: 10.1002/1873-3468.13976

    Ligation of 2′, 3′‐cyclic phosphate miR‐34a‐5p strands to 5′‐OH mRNA mimics in HeLa lysate. (A) Scheme showing principle and different formats of RNA > p ligation. Left: miRNA > p probe bound to a target mRNA inside the RNA‐induced silencing complex (RISC). Right: Alignments of radiolabeled miR‐34a‐5p to LMTK3‐mRNA mimics. (B) Ligation of miR‐34a‐5p > p (22 nt) to different LMTK3 mRNA mimics (64–72 nt) in HeLa lysate. Mir‐34a‐5p > p ligation is shown in lanes 2, 4, 6, 8, and 10. Controls are shown in lanes 11–16. Mir‐34a‐5p > p (2′,3′ cycP +) and miR‐34a‐5p‐3′‐OH (2′,3′ cycP ‐) were 5′‐ 32 P labeled using T4 PNK (3′‐phosphatase minus). Mir‐34a‐5p > p was annealed to different RNA counter‐strands and mixed (8 n m final) with HeLa extract (protein conc. = 2.1 mg·mL −1 ; 0.84 mg·mL −1 final) and a ligation buffer containing KCl (40 n m final), EDTA (pH = 8, 100 µ m final), MgCl 2 (1.2 m m final), DTT (5 m m final), ATP (3 m m final), GTP (0.2 m m final), and RNasin (1 U·µL −1 ; Promega) and incubated at 37 °C for 30 min. In one setup, a bridging ORN b (30 n m , 12 nt) complementarity to each, mir‐34a‐5p > p and LMTK‐3 mimic 8‐8 , was added prior to annealing. Reference oligonucleotides for the respective ligation products (Controls) were chemically synthesized; differences in band intensity may be caused by differences in labeling efficiency. Representative gel shown, n = 3. Full gel shown in Fig. S5 .
    Figure Legend Snippet: Ligation of 2′, 3′‐cyclic phosphate miR‐34a‐5p strands to 5′‐OH mRNA mimics in HeLa lysate. (A) Scheme showing principle and different formats of RNA > p ligation. Left: miRNA > p probe bound to a target mRNA inside the RNA‐induced silencing complex (RISC). Right: Alignments of radiolabeled miR‐34a‐5p to LMTK3‐mRNA mimics. (B) Ligation of miR‐34a‐5p > p (22 nt) to different LMTK3 mRNA mimics (64–72 nt) in HeLa lysate. Mir‐34a‐5p > p ligation is shown in lanes 2, 4, 6, 8, and 10. Controls are shown in lanes 11–16. Mir‐34a‐5p > p (2′,3′ cycP +) and miR‐34a‐5p‐3′‐OH (2′,3′ cycP ‐) were 5′‐ 32 P labeled using T4 PNK (3′‐phosphatase minus). Mir‐34a‐5p > p was annealed to different RNA counter‐strands and mixed (8 n m final) with HeLa extract (protein conc. = 2.1 mg·mL −1 ; 0.84 mg·mL −1 final) and a ligation buffer containing KCl (40 n m final), EDTA (pH = 8, 100 µ m final), MgCl 2 (1.2 m m final), DTT (5 m m final), ATP (3 m m final), GTP (0.2 m m final), and RNasin (1 U·µL −1 ; Promega) and incubated at 37 °C for 30 min. In one setup, a bridging ORN b (30 n m , 12 nt) complementarity to each, mir‐34a‐5p > p and LMTK‐3 mimic 8‐8 , was added prior to annealing. Reference oligonucleotides for the respective ligation products (Controls) were chemically synthesized; differences in band intensity may be caused by differences in labeling efficiency. Representative gel shown, n = 3. Full gel shown in Fig. S5 .

    Techniques Used: Ligation, Labeling, Incubation, Synthesized

    28) Product Images from "Allergen-induced tRNA halves repress focal adhesion of airway smooth muscle cells"

    Article Title: Allergen-induced tRNA halves repress focal adhesion of airway smooth muscle cells

    Journal: bioRxiv

    doi: 10.1101/815803

    Analyses of mRNA- and rRNA-derived cP-RNAs expressed in the lungs of HDM-challenged mice (A) Nucleotide compositions around the 5′- and 3′-ends of mRNA- and rRNA-derived cP-RNAs. A dashed line separates upstream (-) and downstream (+) positions for the 5′- and 3′-ends, representing the cleavage site that generates mRNA-derived cP-RNAs (the regions outside of cP-RNA-generating regions are colored in grey). (B) The alignment patterns of cP-RNAs for the indicated mRNA and rRNA substrates. The position of the three focused mRNA-derived cP-RNAs and two focused rRNA-derived cP-RNAs are shown. (C) Lung total RNAs were treated with CIP or T4 PNK (PNK) and subjected to TaqMan RT-qPCR for the specific quantification of the indicated cP-RNAs. NT designates a non-treated sample used as a negative control. Averages of three independent experiments with SD values are shown. (D) Lung total RNAs control or HDM-challenged mice were subjected to TaqMan RT-qPCR for the indicated cP-RNAs. * P
    Figure Legend Snippet: Analyses of mRNA- and rRNA-derived cP-RNAs expressed in the lungs of HDM-challenged mice (A) Nucleotide compositions around the 5′- and 3′-ends of mRNA- and rRNA-derived cP-RNAs. A dashed line separates upstream (-) and downstream (+) positions for the 5′- and 3′-ends, representing the cleavage site that generates mRNA-derived cP-RNAs (the regions outside of cP-RNA-generating regions are colored in grey). (B) The alignment patterns of cP-RNAs for the indicated mRNA and rRNA substrates. The position of the three focused mRNA-derived cP-RNAs and two focused rRNA-derived cP-RNAs are shown. (C) Lung total RNAs were treated with CIP or T4 PNK (PNK) and subjected to TaqMan RT-qPCR for the specific quantification of the indicated cP-RNAs. NT designates a non-treated sample used as a negative control. Averages of three independent experiments with SD values are shown. (D) Lung total RNAs control or HDM-challenged mice were subjected to TaqMan RT-qPCR for the indicated cP-RNAs. * P

    Techniques Used: Derivative Assay, Mouse Assay, Quantitative RT-PCR, Negative Control

    29) Product Images from "Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities"

    Article Title: Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00484-w

    yUsb1 acts as a 3′–5′exonuclease and CPDase in vitro. a U6 snRNA is synthesized by RNA Polymerase III. Transcription termination produces a heterogeneous U6 with a 4–8 nucleotide U-tail. Processing by yUsb1 shortens the U-tail and leaves a phosphoryl group. b Usb1 removes nucleotides from the 3′ end of RNAs. The 5′-labeled U6 95–112+3U oligonucleotide cis -diol substrate (lane 2) is insensitive to CIP (lane 3) or T4 PNK (lane 4) treatment. Incubation with yUsb1 for 1 h results in a shorter product (lane 5). Similar reactivity of the product to both CIP (lane 6) and T4 PNK (lane 7) indicates that the product is a noncyclic phosphate. An alkaline hydrolysis ladder (lane 1) shows the mobility of oligonucleotide products of different lengths. ( c , top ) One-dimensional 31 P NMR spectra of 2′,3′-cUMP shows a single peak at 20 ppm. A 3′ UMP standard has a single peak at 3.4 ppm. When 2′,3′-cUMP is incubated with AtRNL, which leaves a 2′ phosphate 8 , there is a single peak at 3.2 ppm. Incubation of 2′,3′-cUMP with yUsb1 produces a new signal at 3.4 ppm ( c , bottom ) Zoom of dashed region in top panel. d Time course of Usb1 processing on RNAs with different 3′ end modifications. yUsb1 is most active on RNA substrates with a cis -diol (lanes 1–4), less active on those with a 2′,3′-cyclic phosphate ( > p; lanes 5–8) or 2′ phosphates (2′P; lanes 9–12), and is inactive on 3′ phosphate ends (3′P; lanes 13–16). e Model describing the dual activities of yUsb1
    Figure Legend Snippet: yUsb1 acts as a 3′–5′exonuclease and CPDase in vitro. a U6 snRNA is synthesized by RNA Polymerase III. Transcription termination produces a heterogeneous U6 with a 4–8 nucleotide U-tail. Processing by yUsb1 shortens the U-tail and leaves a phosphoryl group. b Usb1 removes nucleotides from the 3′ end of RNAs. The 5′-labeled U6 95–112+3U oligonucleotide cis -diol substrate (lane 2) is insensitive to CIP (lane 3) or T4 PNK (lane 4) treatment. Incubation with yUsb1 for 1 h results in a shorter product (lane 5). Similar reactivity of the product to both CIP (lane 6) and T4 PNK (lane 7) indicates that the product is a noncyclic phosphate. An alkaline hydrolysis ladder (lane 1) shows the mobility of oligonucleotide products of different lengths. ( c , top ) One-dimensional 31 P NMR spectra of 2′,3′-cUMP shows a single peak at 20 ppm. A 3′ UMP standard has a single peak at 3.4 ppm. When 2′,3′-cUMP is incubated with AtRNL, which leaves a 2′ phosphate 8 , there is a single peak at 3.2 ppm. Incubation of 2′,3′-cUMP with yUsb1 produces a new signal at 3.4 ppm ( c , bottom ) Zoom of dashed region in top panel. d Time course of Usb1 processing on RNAs with different 3′ end modifications. yUsb1 is most active on RNA substrates with a cis -diol (lanes 1–4), less active on those with a 2′,3′-cyclic phosphate ( > p; lanes 5–8) or 2′ phosphates (2′P; lanes 9–12), and is inactive on 3′ phosphate ends (3′P; lanes 13–16). e Model describing the dual activities of yUsb1

    Techniques Used: In Vitro, Synthesized, Labeling, Incubation, Nuclear Magnetic Resonance

    30) Product Images from "Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA"

    Article Title: Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp1163

    Predicted activity of UDG and endoVIII in 454 library preparation of ancient DNA. T4 PNK phosphorylates 5′-ends leaving 5′-phosphate groups. UDG removes uracils, which are concentrated in short 5′- and 3′-overhangs in ancient DNA, leaving abasic sites. EndoVIII then cleaves on both sides of the abasic sites, leaving 5′- and 3′-phosphate groups. T4 polymerase fills in remaining 5′-overhangs and chews back 3′-overhangs, possibly aided by the 3′-phosphatase activity of PNK. Blunt-end ligation and fill-in of sequencing adaptors can then take place.
    Figure Legend Snippet: Predicted activity of UDG and endoVIII in 454 library preparation of ancient DNA. T4 PNK phosphorylates 5′-ends leaving 5′-phosphate groups. UDG removes uracils, which are concentrated in short 5′- and 3′-overhangs in ancient DNA, leaving abasic sites. EndoVIII then cleaves on both sides of the abasic sites, leaving 5′- and 3′-phosphate groups. T4 polymerase fills in remaining 5′-overhangs and chews back 3′-overhangs, possibly aided by the 3′-phosphatase activity of PNK. Blunt-end ligation and fill-in of sequencing adaptors can then take place.

    Techniques Used: Activity Assay, Ancient DNA Assay, Ligation, Sequencing

    31) Product Images from "tRNA-derived fragments and microRNAs in the maternal-fetal interface of a mouse maternal-immune-activation autism model"

    Article Title: tRNA-derived fragments and microRNAs in the maternal-fetal interface of a mouse maternal-immune-activation autism model

    Journal: bioRxiv

    doi: 10.1101/2019.12.20.884650

    Dynamic expression of placental/decidual microRNAs and tRNA fragments. (A) Scheme of sample collection for this figure. Placenta/decidua control samples were collected at embryonic development days E12.5, E13.5, E14.5 and E18.5 (corresponding to the 3 hours, 24 hours, 48 hours, 144 hours time point control samples). (B) Principle component analysis of microRNAs and tRFs from placenta/decidua control samples at different time points by sequence-level analysis. (C) Heatmap shows dynamic expression of placental/decidual microRNAs and tRFs across embryonic development time. Each row in heatmap represents one unique sequence. Expression values were calculated by log10RPM (reads per million total mapped reads) and averaged from 6 samples at each time point and then scaled across row. For visualization purpose, only abundantly expressed miRs or tRFs (mean expression cut-off 100 RPM) that show time-dependent changes are shown in the heatmap. A complete list of dynamically expressed miRs and tRFs please refer to Table S2. (D-E) Examples of dynamically expressed microRNAs. mmu-miR-215-5p (D) is up-regulated over time and mmu-miR-146b-5p (E) is down-regulated over time. (F-H) Examples of dynamically expressed tRFs, including down-regulated 5’ halves from tRNA GluTTC and tRNA GlyCCC . (D-F) Box plots showing RPM (reads per million total mapped reads) for specific miR or tRF sequence, with each dot represents one sample (n = 6 for each time point) and middle line represents median value. (G-H) qRT-PCR validation of temporal decrease of 5’ tRNA halves in both NT (no treatment control) and T4 PNK treatment samples. Error bars represent standard deviation from two biological replicates (male and female control samples pooled for each time point).
    Figure Legend Snippet: Dynamic expression of placental/decidual microRNAs and tRNA fragments. (A) Scheme of sample collection for this figure. Placenta/decidua control samples were collected at embryonic development days E12.5, E13.5, E14.5 and E18.5 (corresponding to the 3 hours, 24 hours, 48 hours, 144 hours time point control samples). (B) Principle component analysis of microRNAs and tRFs from placenta/decidua control samples at different time points by sequence-level analysis. (C) Heatmap shows dynamic expression of placental/decidual microRNAs and tRFs across embryonic development time. Each row in heatmap represents one unique sequence. Expression values were calculated by log10RPM (reads per million total mapped reads) and averaged from 6 samples at each time point and then scaled across row. For visualization purpose, only abundantly expressed miRs or tRFs (mean expression cut-off 100 RPM) that show time-dependent changes are shown in the heatmap. A complete list of dynamically expressed miRs and tRFs please refer to Table S2. (D-E) Examples of dynamically expressed microRNAs. mmu-miR-215-5p (D) is up-regulated over time and mmu-miR-146b-5p (E) is down-regulated over time. (F-H) Examples of dynamically expressed tRFs, including down-regulated 5’ halves from tRNA GluTTC and tRNA GlyCCC . (D-F) Box plots showing RPM (reads per million total mapped reads) for specific miR or tRF sequence, with each dot represents one sample (n = 6 for each time point) and middle line represents median value. (G-H) qRT-PCR validation of temporal decrease of 5’ tRNA halves in both NT (no treatment control) and T4 PNK treatment samples. Error bars represent standard deviation from two biological replicates (male and female control samples pooled for each time point).

    Techniques Used: Expressing, Sequencing, Quantitative RT-PCR, Standard Deviation

    32) Product Images from "Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile"

    Article Title: Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt1009

    Lysidine is absent in tRNA 2 Ile from Bacillus subtilis JJS80 lacking tilS . ( A ) 1D TLC analysis of the wobble position 34 in tRNA 2 Ile purified from B. subtilis wild-type and JJS80. Purified wild-type tRNA 2 and mutant tRNA 2 were partially hydrolyzed by alkali, the 5′ termini of the fragments were 32 P-labeled using T4-PNK. 32 P-labeled fragments were subsequently digested with nuclease P1 and the nature of the 5′ terminal nucleotide was determined by TLC. The solvent used was isobutyric acid:concentrated ammonia:water (66:1:33) ( v : v : v ). The mobility of each nucleotide (pA, pC, pG, pU) was confirmed with non-radiolabeled standards used as internal markers and visualized by UV shadowing. ( B and C ) Template-dependent binding of purified wild-type 3 H-Ile-tRNA 2 (B) and mutant 35 S-Met-tRNA 2 (C) to ribosomes isolated from B. subtilis . Oligonucleotides used were AUG AUA, AUG AUC, AUG AUG, AUG AUU and AUG UUU; the oligonucleotide concentration was 200 μM.
    Figure Legend Snippet: Lysidine is absent in tRNA 2 Ile from Bacillus subtilis JJS80 lacking tilS . ( A ) 1D TLC analysis of the wobble position 34 in tRNA 2 Ile purified from B. subtilis wild-type and JJS80. Purified wild-type tRNA 2 and mutant tRNA 2 were partially hydrolyzed by alkali, the 5′ termini of the fragments were 32 P-labeled using T4-PNK. 32 P-labeled fragments were subsequently digested with nuclease P1 and the nature of the 5′ terminal nucleotide was determined by TLC. The solvent used was isobutyric acid:concentrated ammonia:water (66:1:33) ( v : v : v ). The mobility of each nucleotide (pA, pC, pG, pU) was confirmed with non-radiolabeled standards used as internal markers and visualized by UV shadowing. ( B and C ) Template-dependent binding of purified wild-type 3 H-Ile-tRNA 2 (B) and mutant 35 S-Met-tRNA 2 (C) to ribosomes isolated from B. subtilis . Oligonucleotides used were AUG AUA, AUG AUC, AUG AUG, AUG AUU and AUG UUU; the oligonucleotide concentration was 200 μM.

    Techniques Used: Thin Layer Chromatography, Purification, Mutagenesis, Labeling, Binding Assay, Isolation, Concentration Assay

    Related Articles

    Labeling:

    Article Title: Axl-Gas6 Interaction Counteracts E1A-Mediated Cell Growth Suppression and Proapoptotic Activity
    Article Snippet: Single-stranded cDNA was synthesized with a kit provided by GIBCO-BRL. .. Primer 1 was labeled with [γ-32 P]ATP (Amersham Life Science) and T4 polynucleotide kinase (New England Biolabs) before PCR ( Taq DNA polymerase; Fisher Biotech). .. The RT-PCR products were analyzed by gel electrophoresis with an 8% polyacrylamide gel.

    Article Title: RISC is a 5? phosphomonoester-producing RNA endonuclease
    Article Snippet: .. 5′ labeling reactions contained 10 pmol oligonucleotide, 5 pmol γ-32 PATP (Amersham, 3000 Ci/mmol), 1 unit T4 polynucleotide kinase (New England Biolabs), and 10 mM MgCl2 /5 mM dithiothreitol/70 mM Tris-HCl (pH 7.6) in a final volume of 10 μL. .. The reaction mixture was incubated for 20 min at 37°C, followed by the addition of 1 μL 100 mM ATP and incubation for another 3 min at 37°C.

    Article Title: Nucleobase modification by an RNA enzyme
    Article Snippet: Radiolabeled nucleotides [γ32 P]-ATP, [α32 P]-CTP and [α32 P]-GTP, were purchased from Perkin-Elmer (Waltham, MA). .. Ribozymes were either transcribed using 33nM [α32 P]-CTP or non-radioactive CTP followed by 5΄ end labeled using [γ32 P]-ATP and PNK (NEB). ..

    Polymerase Chain Reaction:

    Article Title: Axl-Gas6 Interaction Counteracts E1A-Mediated Cell Growth Suppression and Proapoptotic Activity
    Article Snippet: Single-stranded cDNA was synthesized with a kit provided by GIBCO-BRL. .. Primer 1 was labeled with [γ-32 P]ATP (Amersham Life Science) and T4 polynucleotide kinase (New England Biolabs) before PCR ( Taq DNA polymerase; Fisher Biotech). .. The RT-PCR products were analyzed by gel electrophoresis with an 8% polyacrylamide gel.

    Amplification:

    Article Title: Tagsteady: a metabarcoding library preparation protocol to avoid false assignment of sequences to samples
    Article Snippet: All library incubations were performed in 0.5 ml Lobind Eppendorf tubes in an Applied Biosystems 2720 thermal cycler. .. End-repair without T4 DNA Polymerase: for treatments −/+ and −/−, an end-repair mastermix was made by combining 4 μl T4 DNA ligase reaction buffer (New England Biolabs, NEB, Ipswich, Massachusetts, US), 0.5 μl dATP (10mM) (Thermo-Fisher), 2 μl reaction booster mix (consisting of 25 % PEG-4000 (Sigma Aldrich, 50%), 2 mg/ml BSA (Thermo-Fisher) and 400 mM NaCl) , 2 μl T4 PNK (NEB, cat#M0201S, 10 U/μl) and 1.5 μl Klenow Fragment (3’- > 5’ exo-) (NEB, cat#M0212S, 5 U/μl) per amplicon pool reaction. .. Ten μl of this mastermix was then added to each 30 μl amplicon pool, mixed well by pipetting and incubated for 30 minutes at 37°C followed by 30 minutes at 65°C and finally cooled to 4°C.

    Ligation:

    Article Title: A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions
    Article Snippet: .. To allow for ligation with the blunt-end of the double-stranded adaptors, 15 µl of purified MNase digested DNA fragments were blunt-ended in a final volume of 20 µl by Klenow fragment of Escherichia coli DNA polymerase (New England Biolabs, CA, USA) and phosphorylated by T4 polynucleotide kinase (New England Biolabs) in a final volume of 30 µl. .. Adaptor ligation The blunt-ended and phosphorylated DNA fragments were ligated to the double-stranded adaptors A and B as described in ( ) with the following modifications.

    Purification:

    Article Title: A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions
    Article Snippet: .. To allow for ligation with the blunt-end of the double-stranded adaptors, 15 µl of purified MNase digested DNA fragments were blunt-ended in a final volume of 20 µl by Klenow fragment of Escherichia coli DNA polymerase (New England Biolabs, CA, USA) and phosphorylated by T4 polynucleotide kinase (New England Biolabs) in a final volume of 30 µl. .. Adaptor ligation The blunt-ended and phosphorylated DNA fragments were ligated to the double-stranded adaptors A and B as described in ( ) with the following modifications.

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    New England Biolabs t4 pnk
    Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, <t>T4</t> PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).
    T4 Pnk, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs dna polymerase i klenow fragment
    Inhibition of Pol I results in <t>DNA</t> damage in a subset of cells a , Representative immunofluorescence images of wild-type and TCOF1 +/− cNCCs stained with an antibody against γH2A.X; quantification is shown in b . c , Representative immunofluorescence images of DNA-damaged wild-type cNCCs stained with an antibody against γH2A.X after 1 h treatment with iPol I or actinomycin D (ActD); quantification is shown in d . e , Representative immunofluorescence images of DNA-damaged HeLa cells stained with an antibody against γH2A.X after 1 h treatment with iPol I; quantification is shown in f . For a – f , cells were collected from n = 3 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. ***P
    Dna Polymerase I Klenow Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, T4 PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).

    Journal: bioRxiv

    Article Title: Tagsteady: a metabarcoding library preparation protocol to avoid false assignment of sequences to samples

    doi: 10.1101/2020.01.22.915009

    Figure Lengend Snippet: Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, T4 PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).

    Article Snippet: End-repair without T4 DNA Polymerase: for treatments −/+ and −/−, an end-repair mastermix was made by combining 4 μl T4 DNA ligase reaction buffer (New England Biolabs, NEB, Ipswich, Massachusetts, US), 0.5 μl dATP (10mM) (Thermo-Fisher), 2 μl reaction booster mix (consisting of 25 % PEG-4000 (Sigma Aldrich, 50%), 2 mg/ml BSA (Thermo-Fisher) and 400 mM NaCl) , 2 μl T4 PNK (NEB, cat#M0201S, 10 U/μl) and 1.5 μl Klenow Fragment (3’- > 5’ exo-) (NEB, cat#M0212S, 5 U/μl) per amplicon pool reaction.

    Techniques: Polymerase Chain Reaction, Activity Assay, Sequencing, Ligation, Amplification

    Effect of arrest on the transcript arrangement in RNAP. ( A ) RNase A footprinting of the transcript in active EC 26 and in arrested EC 27 . The RNA in the complexes was internally labeled at positions +26A or +12C. Each sample was treated with RNase A and fractionated by centrifugation into soluble (s) and matrix-associated (p) fractions before gel analysis. Sequences of the transcripts are shown alongside the autoradiograms, asterisks mark positions of labeling, and arrows show major cuts introduced into the RNA (bold shaded line) by RNase A. The cylinders represent the transcript segments protected by RNAP in the active and arrested complexes. Nonfractionated samples (t) are included as controls. ( B ) 5′-Terminal phosphorylation of the transcripts. RNA-labeled EC 20 and EC 27 (lanes 6 and 4) and arrested fraction of EC 27 purified by chase (lane 2) were treated with T4 polynucleotide kinase in the presence of ATP (lanes 5, 3, and 1). The symbol (P) indicates the phosphorylated transcripts. Arrows indicate the mobility of phosphorylated and nonphosphorylated transcripts.

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

    Article Title: Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3? end of the RNA intact and extruded

    doi:

    Figure Lengend Snippet: Effect of arrest on the transcript arrangement in RNAP. ( A ) RNase A footprinting of the transcript in active EC 26 and in arrested EC 27 . The RNA in the complexes was internally labeled at positions +26A or +12C. Each sample was treated with RNase A and fractionated by centrifugation into soluble (s) and matrix-associated (p) fractions before gel analysis. Sequences of the transcripts are shown alongside the autoradiograms, asterisks mark positions of labeling, and arrows show major cuts introduced into the RNA (bold shaded line) by RNase A. The cylinders represent the transcript segments protected by RNAP in the active and arrested complexes. Nonfractionated samples (t) are included as controls. ( B ) 5′-Terminal phosphorylation of the transcripts. RNA-labeled EC 20 and EC 27 (lanes 6 and 4) and arrested fraction of EC 27 purified by chase (lane 2) were treated with T4 polynucleotide kinase in the presence of ATP (lanes 5, 3, and 1). The symbol (P) indicates the phosphorylated transcripts. Arrows indicate the mobility of phosphorylated and nonphosphorylated transcripts.

    Article Snippet: EC11 (the numerical index denotes the length of the transcript) was treated with 10 units of T4 polynucleotide kinase (New England Biolabs) and 50 μCi of [α-32 P]ATP (4500 Ci/mmol; ICN Biomedicals, Costa Mesa, CA) for 10 min to label the DNA, washed with TB, and walked to the desired position.

    Techniques: Footprinting, Labeling, Centrifugation, Purification

    Inhibition of Pol I results in DNA damage in a subset of cells a , Representative immunofluorescence images of wild-type and TCOF1 +/− cNCCs stained with an antibody against γH2A.X; quantification is shown in b . c , Representative immunofluorescence images of DNA-damaged wild-type cNCCs stained with an antibody against γH2A.X after 1 h treatment with iPol I or actinomycin D (ActD); quantification is shown in d . e , Representative immunofluorescence images of DNA-damaged HeLa cells stained with an antibody against γH2A.X after 1 h treatment with iPol I; quantification is shown in f . For a – f , cells were collected from n = 3 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. ***P

    Journal: Nature

    Article Title: Tissue–selective effects of nucleolar stress and rDNA damage in developmental disorders

    doi: 10.1038/nature25449

    Figure Lengend Snippet: Inhibition of Pol I results in DNA damage in a subset of cells a , Representative immunofluorescence images of wild-type and TCOF1 +/− cNCCs stained with an antibody against γH2A.X; quantification is shown in b . c , Representative immunofluorescence images of DNA-damaged wild-type cNCCs stained with an antibody against γH2A.X after 1 h treatment with iPol I or actinomycin D (ActD); quantification is shown in d . e , Representative immunofluorescence images of DNA-damaged HeLa cells stained with an antibody against γH2A.X after 1 h treatment with iPol I; quantification is shown in f . For a – f , cells were collected from n = 3 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. ***P

    Article Snippet: After the NT2 wash, DDX21-bound RNA–protein complexes were dephosphorylated with T4 PNK (NEB, catalogue number M0210) for 30 min in an Eppendorf Thermomixer at 37 °C, 15 s at 1,400 r.p.m., 90 s rest in a 30 μl reaction, pH 6.5, containing 10 units of T4 PNK, 0.1 μl SUPERase-IN, and 6 μl of PEG-400 (16.7% final).

    Techniques: Inhibition, Immunofluorescence, Staining