lpp 21 primer  (New England Biolabs)


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    Structured Review

    New England Biolabs lpp 21 primer
    Txe-induced cleavage of  lpp  mRNA. The 5′ end of  lpp  mRNA was mapped by using the primer  lpp  21. Numbers indicate times (min) at which mRNA was harvested after the addition of IPTG. The major cleavage site is indicated by an arrow. The sequence around the major cleavage site is shown to the side, with the cleavage site again indicated by an arrow.
    Lpp 21 Primer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 1913 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lpp 21 primer/product/New England Biolabs
    Average 85 stars, based on 1913 article reviews
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    lpp 21 primer - by Bioz Stars, 2020-07
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    Images

    1) Product Images from "Txe, an endoribonuclease of the enterococcal Axe-Txe toxin-antitoxin system, cleaves mRNA and inhibits protein synthesis"

    Article Title: Txe, an endoribonuclease of the enterococcal Axe-Txe toxin-antitoxin system, cleaves mRNA and inhibits protein synthesis

    Journal: Microbiology

    doi: 10.1099/mic.0.045492-0

    Txe-induced cleavage of  lpp  mRNA. The 5′ end of  lpp  mRNA was mapped by using the primer  lpp  21. Numbers indicate times (min) at which mRNA was harvested after the addition of IPTG. The major cleavage site is indicated by an arrow. The sequence around the major cleavage site is shown to the side, with the cleavage site again indicated by an arrow.
    Figure Legend Snippet: Txe-induced cleavage of lpp mRNA. The 5′ end of lpp mRNA was mapped by using the primer lpp 21. Numbers indicate times (min) at which mRNA was harvested after the addition of IPTG. The major cleavage site is indicated by an arrow. The sequence around the major cleavage site is shown to the side, with the cleavage site again indicated by an arrow.

    Techniques Used: Sequencing

    2) Product Images from "Modulation of telomerase activity by telomere DNA-binding proteins in Oxytricha"

    Article Title: Modulation of telomerase activity by telomere DNA-binding proteins in Oxytricha

    Journal: Genes & Development

    doi:

    Cross-linking of the α subunit to the DNA substrate (T 4 G 4 ) 2 . ( A ) To monitor complex formation, the DNA was 5′-radiolabeled, incubated with the α subunit, and cross-linked. Cross-linked complexes were monitored by denaturing polyacrylamide gel electrophoresis. ( B ) To evaluate whether the α–DNA and α–DNA–α cross-linked complexes were extended by telomerase, increasing concentrations of the α subunit were cross-linked to nonlabeled DNA. 32 P-Radiolabeled nucleotides and nuclear extract were added, and the label incorporation into the complexes was monitored by gel electrophoresis.
    Figure Legend Snippet: Cross-linking of the α subunit to the DNA substrate (T 4 G 4 ) 2 . ( A ) To monitor complex formation, the DNA was 5′-radiolabeled, incubated with the α subunit, and cross-linked. Cross-linked complexes were monitored by denaturing polyacrylamide gel electrophoresis. ( B ) To evaluate whether the α–DNA and α–DNA–α cross-linked complexes were extended by telomerase, increasing concentrations of the α subunit were cross-linked to nonlabeled DNA. 32 P-Radiolabeled nucleotides and nuclear extract were added, and the label incorporation into the complexes was monitored by gel electrophoresis.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis, Nucleic Acid Electrophoresis

    Telomere protein modulation of telomerase: a model for telomere length regulation. ( A ) The effects of telomere protein subunits on telomerase elongation of telomeric DNA are summarized schematically. The α subunit is light gray and hatched, whereas the β subunit is a darker gray and striped. ( B ) A proposed model for the in vivo telomere protein involvement in telomere length regulation. Exactly when telomere proteins bind relative to telomerase extension is speculative.
    Figure Legend Snippet: Telomere protein modulation of telomerase: a model for telomere length regulation. ( A ) The effects of telomere protein subunits on telomerase elongation of telomeric DNA are summarized schematically. The α subunit is light gray and hatched, whereas the β subunit is a darker gray and striped. ( B ) A proposed model for the in vivo telomere protein involvement in telomere length regulation. Exactly when telomere proteins bind relative to telomerase extension is speculative.

    Techniques Used: In Vivo

    3) Product Images from "Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis"

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky067

    Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).
    Figure Legend Snippet: Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).

    Techniques Used: Plasmid Preparation, Isolation, Purification, Polymerase Chain Reaction, Clone Assay

    4) Product Images from "Single-molecule analysis of DNA replication in Xenopus egg extracts"

    Article Title: Single-molecule analysis of DNA replication in Xenopus egg extracts

    Journal: Methods (San Diego, Calif.)

    doi: 10.1016/j.ymeth.2012.03.033

    Replication of surface-immobilized λ DNA in Xenopus ].
    Figure Legend Snippet: Replication of surface-immobilized λ DNA in Xenopus ].

    Techniques Used:

    5) Product Images from "Towards the Generation of an ASFV-pA104R DISC Mutant and a Complementary Cell Line—A Potential Methodology for the Production of a Vaccine Candidate"

    Article Title: Towards the Generation of an ASFV-pA104R DISC Mutant and a Complementary Cell Line—A Potential Methodology for the Production of a Vaccine Candidate

    Journal: Vaccines

    doi: 10.3390/vaccines7030068

    Open reading frame (ORF) A104R was successfully integrated in transfected Vero and COS-1 cells. ORF A104R was amplified by PCR (287 bp) from the genome of Vero-pA104R clones, cultured under antibiotic selective pressure (G418). Line 1: Molecular marker (NZYDNA Ladder III); Line 2: Vero wild type (wt, negative control); Line 3: pIRESneo A104R (positive control); Lines 4–6: three distinct Vero-pA104R clones, transfected with linearized plasmid and Lipofectamine 2000; Lines 7,8: two distinct Vero-pA104R clones, transfected with circular plasmid and Lipofectamine 2000; Line 9: COS-1 wt cells (negative control); Line 10: COS-1 cells transfected by Lipofectamine 2000 with the A104R construct; Line 11: Flp-In wt cells; Line 12: Flp-In cells transformed with the A104R construct; Lines 13–15: COS-1 cells transfected with Lipofectamine LTX (16 weeks); Line 16: FRT/A104R plasmid (positive control); Lines 17–20: Flp-In clones sustaining the selective pressure.
    Figure Legend Snippet: Open reading frame (ORF) A104R was successfully integrated in transfected Vero and COS-1 cells. ORF A104R was amplified by PCR (287 bp) from the genome of Vero-pA104R clones, cultured under antibiotic selective pressure (G418). Line 1: Molecular marker (NZYDNA Ladder III); Line 2: Vero wild type (wt, negative control); Line 3: pIRESneo A104R (positive control); Lines 4–6: three distinct Vero-pA104R clones, transfected with linearized plasmid and Lipofectamine 2000; Lines 7,8: two distinct Vero-pA104R clones, transfected with circular plasmid and Lipofectamine 2000; Line 9: COS-1 wt cells (negative control); Line 10: COS-1 cells transfected by Lipofectamine 2000 with the A104R construct; Line 11: Flp-In wt cells; Line 12: Flp-In cells transformed with the A104R construct; Lines 13–15: COS-1 cells transfected with Lipofectamine LTX (16 weeks); Line 16: FRT/A104R plasmid (positive control); Lines 17–20: Flp-In clones sustaining the selective pressure.

    Techniques Used: Transfection, Amplification, Polymerase Chain Reaction, Clone Assay, Cell Culture, Marker, Negative Control, Positive Control, Plasmid Preparation, Construct, Transformation Assay

    6) Product Images from "Detecting RNA-RNA interactions in E. coli using a modified CLASH method"

    Article Title: Detecting RNA-RNA interactions in E. coli using a modified CLASH method

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-3725-3

    Schematic overview of the modified protocol. a , wet experiment. Irradiated with 365 nm UV, RNAs were cross-linked by AMT at the paired region, and survive DNase I, RNase T1 and RNase H treatments which digest DNA and single strand RNA. Cross-linked RNAs were ligated by T4 RNA ligase 1. After photoreversal of cross-linkages by 254 nm UV, the ligated RNAs could be sequenced and identified. b , bioinformatics analysis
    Figure Legend Snippet: Schematic overview of the modified protocol. a , wet experiment. Irradiated with 365 nm UV, RNAs were cross-linked by AMT at the paired region, and survive DNase I, RNase T1 and RNase H treatments which digest DNA and single strand RNA. Cross-linked RNAs were ligated by T4 RNA ligase 1. After photoreversal of cross-linkages by 254 nm UV, the ligated RNAs could be sequenced and identified. b , bioinformatics analysis

    Techniques Used: Modification, Irradiation

    7) Product Images from "Multistep assembly of DNA condensation clusters by SMC"

    Article Title: Multistep assembly of DNA condensation clusters by SMC

    Journal: Nature Communications

    doi: 10.1038/ncomms10200

    Characterization of BsSMC sliding along DNA. ( a ) Cartoon of the BsSMC dimer. The Walker A motif resides on the N-terminal half of the head, while Walker B and C motifs are located at the C terminal part of the head. Not drawn to scale. ( b ) Snapshots from two different DNAs showing diffusion of labelled wild-type BsSMC (∼2 nM) on a flow-stretched DNA in the presence of 1 mM ATP. White and black scale bars, 5 s and 3 μm, respectively. ( c ) Trajectories of BsSMC in the presence of 1 mM ATP. Different events were denoted with different colours, and the starting point of each event was arbitrarily set for visualization on the same plot. ( d ) Two trajectories of wild-type BsSMC showing transitions between static association and 1D diffusion in the presence of 1 mM ATP. Light-brown boxes represent periods of static binding as defined by the criteria described in the text. ( e ) A histogram of s.d. of statically bound BsSMC position in the transverse direction ( n =22). Light-yellow and green colours denote approximate regions of the proximal and distal halves of the λ-DNA, respectively. This distinction was determined based on the characterization of transverse fluctuation along the DNA length ( Supplementary Fig. 1e ). ( f ) Histograms of net displacement. Positive values represent net displacement towards the free end of the DNA. A dotted vertical green line represents zero net displacement. Horizontal blue ( n =25) and red ( n =33) bars in the histogram represent data without and with ATP, respectively ( P > 0.2 by t -test). The horizontal blue (without ATP) and red (with ATP) lines on the top of the graph represent 95% confidence intervals obtained by bootstrapping analyses. ( g ) Histograms for diffusion coefficients of wild-type BsSMC. n =19 for blue bars, n =21 for red bars ( P > 0.2 by t -test). The horizontal blue (without ATP) and red (with ATP) lines on the top of the graph correspond to 95% confidence intervals from bootstrapping analysis.
    Figure Legend Snippet: Characterization of BsSMC sliding along DNA. ( a ) Cartoon of the BsSMC dimer. The Walker A motif resides on the N-terminal half of the head, while Walker B and C motifs are located at the C terminal part of the head. Not drawn to scale. ( b ) Snapshots from two different DNAs showing diffusion of labelled wild-type BsSMC (∼2 nM) on a flow-stretched DNA in the presence of 1 mM ATP. White and black scale bars, 5 s and 3 μm, respectively. ( c ) Trajectories of BsSMC in the presence of 1 mM ATP. Different events were denoted with different colours, and the starting point of each event was arbitrarily set for visualization on the same plot. ( d ) Two trajectories of wild-type BsSMC showing transitions between static association and 1D diffusion in the presence of 1 mM ATP. Light-brown boxes represent periods of static binding as defined by the criteria described in the text. ( e ) A histogram of s.d. of statically bound BsSMC position in the transverse direction ( n =22). Light-yellow and green colours denote approximate regions of the proximal and distal halves of the λ-DNA, respectively. This distinction was determined based on the characterization of transverse fluctuation along the DNA length ( Supplementary Fig. 1e ). ( f ) Histograms of net displacement. Positive values represent net displacement towards the free end of the DNA. A dotted vertical green line represents zero net displacement. Horizontal blue ( n =25) and red ( n =33) bars in the histogram represent data without and with ATP, respectively ( P > 0.2 by t -test). The horizontal blue (without ATP) and red (with ATP) lines on the top of the graph represent 95% confidence intervals obtained by bootstrapping analyses. ( g ) Histograms for diffusion coefficients of wild-type BsSMC. n =19 for blue bars, n =21 for red bars ( P > 0.2 by t -test). The horizontal blue (without ATP) and red (with ATP) lines on the top of the graph correspond to 95% confidence intervals from bootstrapping analysis.

    Techniques Used: Diffusion-based Assay, Flow Cytometry, Binding Assay

    8) 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

    9) 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

    10) 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

    11) Product Images from "Cell Cycle-Regulated Expression of Mammalian CDC6 Is Dependent on E2F"

    Article Title: Cell Cycle-Regulated Expression of Mammalian CDC6 Is Dependent on E2F

    Journal: Molecular and Cellular Biology

    doi:

    In vivo footprinting analysis of the transcription start site region of the hCDC6 promoter. LMPCRs were performed with primer S1, S2, or [minus (−) strand] or AS1, AS2, or AS3 [plus (+) strand] on genomic DNA templates obtained from serum-starved (G 0 ) or exponentionally growing (expo.) MCF7 cells treated in vivo with the guanosine methylating agent, DMS. Similar LMPCRs were performed with DMS-methylated naked DNA (vitro lanes). Protected residues and hyperreactive residues detected between in vitro- and in vivo-methylated DNAs are indicated as circles and arrowheads, respectively. Weak (white circles) and strong (black circles) in vivo protection is indicated. The transcription start site is indicated with an arrowhead (+1) to the left of the blots. Amplified DNA ladders that are visible correspond to guanines of the hCDC6 promoter. (A) Positive-sense strand. (B) Negative-sense strand. (C) Summary of DNA-protein contacts observed by in vivo footprinting on both strands of the hCDC6 promoter upstream of the transcription start site (black arrow, +1). Putative consensus binding sites are indicated as open boxes. A protein-bound element with a sequence similar to that of an Sp1 consensus site is depicted as Sp1/?. A protected site around the putative initiator region (INR) (bp −16 to −10) is indicated with a question mark.
    Figure Legend Snippet: In vivo footprinting analysis of the transcription start site region of the hCDC6 promoter. LMPCRs were performed with primer S1, S2, or [minus (−) strand] or AS1, AS2, or AS3 [plus (+) strand] on genomic DNA templates obtained from serum-starved (G 0 ) or exponentionally growing (expo.) MCF7 cells treated in vivo with the guanosine methylating agent, DMS. Similar LMPCRs were performed with DMS-methylated naked DNA (vitro lanes). Protected residues and hyperreactive residues detected between in vitro- and in vivo-methylated DNAs are indicated as circles and arrowheads, respectively. Weak (white circles) and strong (black circles) in vivo protection is indicated. The transcription start site is indicated with an arrowhead (+1) to the left of the blots. Amplified DNA ladders that are visible correspond to guanines of the hCDC6 promoter. (A) Positive-sense strand. (B) Negative-sense strand. (C) Summary of DNA-protein contacts observed by in vivo footprinting on both strands of the hCDC6 promoter upstream of the transcription start site (black arrow, +1). Putative consensus binding sites are indicated as open boxes. A protein-bound element with a sequence similar to that of an Sp1 consensus site is depicted as Sp1/?. A protected site around the putative initiator region (INR) (bp −16 to −10) is indicated with a question mark.

    Techniques Used: In Vivo, Footprinting, Methylation, In Vitro, Amplification, Binding Assay, Sequencing

    12) 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

    13) Product Images from "Crosslinking of Ribosomal Proteins to RNA in Maize Ribosomes by UV-B and Its Effects on Translation 1Crosslinking of Ribosomal Proteins to RNA in Maize Ribosomes by UV-B and Its Effects on Translation 1 [w]"

    Article Title: Crosslinking of Ribosomal Proteins to RNA in Maize Ribosomes by UV-B and Its Effects on Translation 1Crosslinking of Ribosomal Proteins to RNA in Maize Ribosomes by UV-B and Its Effects on Translation 1 [w]

    Journal: Plant Physiology

    doi: 10.1104/pp.104.047043

    Crosslinking of ribosomal proteins and RNA in a dosage-dependent manner. A and D, Autoradiography of [ 32 P]-labeled RNA crosslinked to ribosomal proteins; B and E, Coomassie Blue-stained gel. A and B, After UV-B exposure of leaves for 2, 4, 6, and 8 h, purified ribosomes were treated as described in “Materials and Methods.” Recovery: After 8 h of UV-B, leaves were maintained without UV-B for 4 or 16 h in the dark. D and E, Controls: Proteins were incubated with DNAse I or with proteinase K after the treatment with RNases; the labeling reaction was done without T4 polynucleotide kinase (−T4 PNK); and yeast RNA was used instead of purified ribosomes. C, Quantification of radioactive bands by densitometry of the autoradiograph. The percentage of labeling was corrected for loading differences per lane. The molecular mass of marker proteins is indicated in the right of A and B. Figure 1A shows the result of one experiment, the same bands were detected in all eight experiments.
    Figure Legend Snippet: Crosslinking of ribosomal proteins and RNA in a dosage-dependent manner. A and D, Autoradiography of [ 32 P]-labeled RNA crosslinked to ribosomal proteins; B and E, Coomassie Blue-stained gel. A and B, After UV-B exposure of leaves for 2, 4, 6, and 8 h, purified ribosomes were treated as described in “Materials and Methods.” Recovery: After 8 h of UV-B, leaves were maintained without UV-B for 4 or 16 h in the dark. D and E, Controls: Proteins were incubated with DNAse I or with proteinase K after the treatment with RNases; the labeling reaction was done without T4 polynucleotide kinase (−T4 PNK); and yeast RNA was used instead of purified ribosomes. C, Quantification of radioactive bands by densitometry of the autoradiograph. The percentage of labeling was corrected for loading differences per lane. The molecular mass of marker proteins is indicated in the right of A and B. Figure 1A shows the result of one experiment, the same bands were detected in all eight experiments.

    Techniques Used: Autoradiography, Labeling, Staining, Purification, Incubation, Marker

    14) 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

    15) Product Images from "Mapping the Human miRNA Interactome by CLASH Reveals Frequent Noncanonical Binding"

    Article Title: Mapping the Human miRNA Interactome by CLASH Reveals Frequent Noncanonical Binding

    Journal: Cell

    doi: 10.1016/j.cell.2013.03.043

    Overview of Experimental and Bioinformatic Procedures (A) Growing cells were UV irradiated, and PTH-AGO1 was purified. RNA fragmentation, ligation, cDNA synthesis, and sequencing of AGO1-associated RNAs allowed the identification of sites of AGO1 binding (as single reads) and RNA-RNA interactions at AGO1-binding sites (as chimeric reads). (B) Sequencing reads were mapped to a database of human transcripts using BLAST ( Altschul et al., 1990 ). Sequences reliably mapped to two different sites were folded in silico using UNAFold ( Markham and Zuker, 2008 ) to identify the interaction site of the RNA molecules that gave rise to the chimeric cDNA. (C) Example interaction between miR-196a/b and HOXC8 that was supported by chimeric reads (red), and a cluster of nonchimeric reads (green). The blue dashed line represents the location of the miRNA bit of chimera, and the red dashed line shows the 25 nt mRNA extension added during the analysis. The interaction was previously shown experimentally ( Li et al., 2010 ) and can be predicted by RNAhybrid ( Rehmsmeier et al., 2004 ). (D) Distribution of all miRNA interactions among various classes of RNAs. The main miRNA targets are mRNAs and are represented by 18,514 interactions. See also Figure S1 and Tables S1 and S2 A–S2C.
    Figure Legend Snippet: Overview of Experimental and Bioinformatic Procedures (A) Growing cells were UV irradiated, and PTH-AGO1 was purified. RNA fragmentation, ligation, cDNA synthesis, and sequencing of AGO1-associated RNAs allowed the identification of sites of AGO1 binding (as single reads) and RNA-RNA interactions at AGO1-binding sites (as chimeric reads). (B) Sequencing reads were mapped to a database of human transcripts using BLAST ( Altschul et al., 1990 ). Sequences reliably mapped to two different sites were folded in silico using UNAFold ( Markham and Zuker, 2008 ) to identify the interaction site of the RNA molecules that gave rise to the chimeric cDNA. (C) Example interaction between miR-196a/b and HOXC8 that was supported by chimeric reads (red), and a cluster of nonchimeric reads (green). The blue dashed line represents the location of the miRNA bit of chimera, and the red dashed line shows the 25 nt mRNA extension added during the analysis. The interaction was previously shown experimentally ( Li et al., 2010 ) and can be predicted by RNAhybrid ( Rehmsmeier et al., 2004 ). (D) Distribution of all miRNA interactions among various classes of RNAs. The main miRNA targets are mRNAs and are represented by 18,514 interactions. See also Figure S1 and Tables S1 and S2 A–S2C.

    Techniques Used: Irradiation, Purification, Ligation, Sequencing, Binding Assay, In Silico

    16) Product Images from "Arginine methylation of DRBD18 differentially impacts its opposing effects on the trypanosome transcriptome"

    Article Title: Arginine methylation of DRBD18 differentially impacts its opposing effects on the trypanosome transcriptome

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv428

    Effects of hypomethylation and methylmimic on subcellular localization and RNA binding capacity of DRBD18. ( A ) Biochemical fractionation and western blot analysis of MHT-DRBD18(WT), MHT-DRBD18(R→K) and MHT-DRBD18(R→F) expressing cell lines. Cells were harvested two days post-induction and 1 × 10 6 cellular equivalents of cytoplasmic and nuclear fractions were separated via 10% SDS-PAGE. HSP70 and NOG are cytoplasmic and nuclear markers, respectively. MHT-DRBD18 constructs were visualized using anti-myc antibody. W, whole cell; C, cytoplasmic fraction; N, nuclear fraction. ( B ) Indirect immunofluorescence of uninduced MHT-DRBD18(WT) expressing cells and MHT-DRBD18(WT), MHT-DRBD18(R→K), and MHT-DRBD18(R→F) expressing cell lines 2 days post-induction with tet using anti-myc antibody. Nuclei and kinetoplasts were stained with DAPI, and merged signals (myc/DAPI) are shown on the right. DIC, differential interference contrast. Inset shows higher magnification image of perinuclear labeling. ( C ) CLIP analysis of cells expressing MHT-DRBD18 variants. Cells expressing MHT-DRBD18 variants were UV crosslinked at 254 nm and proteins were precipitated using anti-myc conjugated resin. Cell lysate pooled from all three samples and incubated with uncoated beads served as a negative control sample (mock). Left panel shows anti-myc western blot analysis of input samples (7.5 × 10 5 cell equivalents). Also shown are immunoprecipitated (bound) MHT-DRDB18 variant proteins following in vivo crosslinking and immunoprecipitation and just prior to labeling with γ 32 P-ATP by polynucleotide kinase. These bound samples were used for the analyses shown in the right panel. I, input, B, bound samples. Right panel shows phosphorimage analysis of bound MHT-DRBD18 variant RNPs. Values below the image represent the average signals from three replicate experiments.
    Figure Legend Snippet: Effects of hypomethylation and methylmimic on subcellular localization and RNA binding capacity of DRBD18. ( A ) Biochemical fractionation and western blot analysis of MHT-DRBD18(WT), MHT-DRBD18(R→K) and MHT-DRBD18(R→F) expressing cell lines. Cells were harvested two days post-induction and 1 × 10 6 cellular equivalents of cytoplasmic and nuclear fractions were separated via 10% SDS-PAGE. HSP70 and NOG are cytoplasmic and nuclear markers, respectively. MHT-DRBD18 constructs were visualized using anti-myc antibody. W, whole cell; C, cytoplasmic fraction; N, nuclear fraction. ( B ) Indirect immunofluorescence of uninduced MHT-DRBD18(WT) expressing cells and MHT-DRBD18(WT), MHT-DRBD18(R→K), and MHT-DRBD18(R→F) expressing cell lines 2 days post-induction with tet using anti-myc antibody. Nuclei and kinetoplasts were stained with DAPI, and merged signals (myc/DAPI) are shown on the right. DIC, differential interference contrast. Inset shows higher magnification image of perinuclear labeling. ( C ) CLIP analysis of cells expressing MHT-DRBD18 variants. Cells expressing MHT-DRBD18 variants were UV crosslinked at 254 nm and proteins were precipitated using anti-myc conjugated resin. Cell lysate pooled from all three samples and incubated with uncoated beads served as a negative control sample (mock). Left panel shows anti-myc western blot analysis of input samples (7.5 × 10 5 cell equivalents). Also shown are immunoprecipitated (bound) MHT-DRDB18 variant proteins following in vivo crosslinking and immunoprecipitation and just prior to labeling with γ 32 P-ATP by polynucleotide kinase. These bound samples were used for the analyses shown in the right panel. I, input, B, bound samples. Right panel shows phosphorimage analysis of bound MHT-DRBD18 variant RNPs. Values below the image represent the average signals from three replicate experiments.

    Techniques Used: RNA Binding Assay, Fractionation, Western Blot, Expressing, SDS Page, Construct, Immunofluorescence, Staining, Labeling, Cross-linking Immunoprecipitation, Incubation, Negative Control, Immunoprecipitation, Variant Assay, In Vivo

    17) Product Images from "New CRISPR-Cas systems from uncultivated microbes"

    Article Title: New CRISPR-Cas systems from uncultivated microbes

    Journal: Nature

    doi: 10.1038/nature21059

    CRISPR-CasX is a dual-guided system that mediates programmable DNA interference in E. coli a , Diagram of CasX plasmid interference assays. b , Serial dilution of E. coli expressing the Planctomycetes CasX locus with spacer 1 (sX1) and transformed with the specified target (sX1, CasX protospacer 1; sX2, CasX protospacer 2; NT, non-target). c , Plasmid interference by Deltaproteobacteria CasX, using the same spacers and targets as in ( b ). d , PAM depletion assays for the Planctomycetes CasX locus expressed in E. coli . Sequence logo was generated from PAM sequences depleted > 30-fold compared to a control library (see also Extended Data Fig. 8 ). e , Diagram of CasX DNA interference. f , Mapping of environmental RNA sequences to the CasX CRISPR locus (red arrow, putative tracrRNA; white boxes, repeats; green diamonds, spacers); Inset: detailed view of mapping to first repeat and spacer. g , Plasmid interference assays with the putative tracrRNA knocked out of the CasX locus and CasX coexpressed with a crRNA alone, a truncated sgRNA or a full length sgRNA (T, target; NT, non-target). Experiments presented in ( c ) and ( g ) were conducted in triplicate and mean ± s.d. is shown.
    Figure Legend Snippet: CRISPR-CasX is a dual-guided system that mediates programmable DNA interference in E. coli a , Diagram of CasX plasmid interference assays. b , Serial dilution of E. coli expressing the Planctomycetes CasX locus with spacer 1 (sX1) and transformed with the specified target (sX1, CasX protospacer 1; sX2, CasX protospacer 2; NT, non-target). c , Plasmid interference by Deltaproteobacteria CasX, using the same spacers and targets as in ( b ). d , PAM depletion assays for the Planctomycetes CasX locus expressed in E. coli . Sequence logo was generated from PAM sequences depleted > 30-fold compared to a control library (see also Extended Data Fig. 8 ). e , Diagram of CasX DNA interference. f , Mapping of environmental RNA sequences to the CasX CRISPR locus (red arrow, putative tracrRNA; white boxes, repeats; green diamonds, spacers); Inset: detailed view of mapping to first repeat and spacer. g , Plasmid interference assays with the putative tracrRNA knocked out of the CasX locus and CasX coexpressed with a crRNA alone, a truncated sgRNA or a full length sgRNA (T, target; NT, non-target). Experiments presented in ( c ) and ( g ) were conducted in triplicate and mean ± s.d. is shown.

    Techniques Used: CRISPR, Plasmid Preparation, Serial Dilution, Expressing, Transformation Assay, Sequencing, Generated

    Programmed DNA interference by CasX a , Plasmid interference assays for CasX.1 (Deltaproteobacteria) and CasX.2 (Planctomycetes), continued from Figure 3c (sX1, CasX spacer 1; sX2, CasX spacer 2; NT, non-target). Experiments were conducted in triplicate and mean ± s.d. is shown. b , Serial dilution of E. coli expressing a CasX locus and transformed with the specified target, continued from Figure 3b . c , PAM depletion assays for the Deltaproteobacteria CasX and d , Planctomycetes CasX expressed in E. coli. PAM sequences depleted greater than the indicated PAM depletion value threshold (PDVT) compared to a control library were used to generate the sequence logo. e , Diagram depicting the location of Northern blot probes for CasX.1. f , Northern blots for CasX.1 tracrRNA in total RNA extracted from E. coli expressing the CasX.1 locus. The sequences of the probes used are provided in Supplementary Table 2.
    Figure Legend Snippet: Programmed DNA interference by CasX a , Plasmid interference assays for CasX.1 (Deltaproteobacteria) and CasX.2 (Planctomycetes), continued from Figure 3c (sX1, CasX spacer 1; sX2, CasX spacer 2; NT, non-target). Experiments were conducted in triplicate and mean ± s.d. is shown. b , Serial dilution of E. coli expressing a CasX locus and transformed with the specified target, continued from Figure 3b . c , PAM depletion assays for the Deltaproteobacteria CasX and d , Planctomycetes CasX expressed in E. coli. PAM sequences depleted greater than the indicated PAM depletion value threshold (PDVT) compared to a control library were used to generate the sequence logo. e , Diagram depicting the location of Northern blot probes for CasX.1. f , Northern blots for CasX.1 tracrRNA in total RNA extracted from E. coli expressing the CasX.1 locus. The sequences of the probes used are provided in Supplementary Table 2.

    Techniques Used: Plasmid Preparation, Serial Dilution, Expressing, Transformation Assay, Sequencing, Northern Blot

    18) Product Images from "Bacteriophage DNA glucosylation impairs target DNA binding by type I and II but not by type V CRISPR–Cas effector complexes"

    Article Title: Bacteriophage DNA glucosylation impairs target DNA binding by type I and II but not by type V CRISPR–Cas effector complexes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1264

    Effect of T4 DNA modifications on type II-A CRISPR–Cas sgRNA mediated DNA targeting. ( A ) Schematic of DNA targeting by Cas9. Modified cytosine residues are indicated in red. Cleavage sites are indicated by black arrows. ( B ) Cleavage assay of Cas9 on 98 bp modified targets (indicated by black arrow). Cas9 is loaded with either targeting sgRNA (T sgRNA) or non-targeting sgRNA (NT sgRNA). Restriction products of Cas9 are 61 and 37 bp. ( C ) EMSA of dCas9 on target DNA containing C, 5-hmC or 5-ghmC (indicated by black arrow) at increasing protein concentrations [nM]. Fraction of bound target is indicated by white arrows.
    Figure Legend Snippet: Effect of T4 DNA modifications on type II-A CRISPR–Cas sgRNA mediated DNA targeting. ( A ) Schematic of DNA targeting by Cas9. Modified cytosine residues are indicated in red. Cleavage sites are indicated by black arrows. ( B ) Cleavage assay of Cas9 on 98 bp modified targets (indicated by black arrow). Cas9 is loaded with either targeting sgRNA (T sgRNA) or non-targeting sgRNA (NT sgRNA). Restriction products of Cas9 are 61 and 37 bp. ( C ) EMSA of dCas9 on target DNA containing C, 5-hmC or 5-ghmC (indicated by black arrow) at increasing protein concentrations [nM]. Fraction of bound target is indicated by white arrows.

    Techniques Used: CRISPR, Modification, Cleavage Assay

    Effect of T4 DNA modifications of PAM-complementary cytosines on type II-A CRISPR–Cas sgRNA mediated DNA targeting. Cleavage assay of Cas9 on target DNA containing 5-hmC (indicated in red). Cas9 is loaded with either targeting sgRNA (T sgRNA) or non-targeting sgRNA (NT sgRNA). Restriction products of Cas9 are 57 and 33 bp. The marker is indicated by white arrows.
    Figure Legend Snippet: Effect of T4 DNA modifications of PAM-complementary cytosines on type II-A CRISPR–Cas sgRNA mediated DNA targeting. Cleavage assay of Cas9 on target DNA containing 5-hmC (indicated in red). Cas9 is loaded with either targeting sgRNA (T sgRNA) or non-targeting sgRNA (NT sgRNA). Restriction products of Cas9 are 57 and 33 bp. The marker is indicated by white arrows.

    Techniques Used: CRISPR, Cleavage Assay, Marker

    Effect of T4 DNA modifications on type I-E CRISPR–Cas sgRNA mediated DNA targeting. ( A ) Schematic of DNA targeting and R-loop formation by Cascade. Modified cytosine residues are indicated in red. ( B ) Cleavage assay of Cas3 in conjunction with Cascade on 98 bp modified targets, indicated by black arrow. The marker is indicated by white arrows. Cascade effector complexes are loaded with either targeting crRNA (T crRNA) or non-targeting crRNA (NT crRNA). Restriction products of Cas3 are of undefined length. ( C ). Electrophoretic Mobility Shift Assay (EMSA) of Cascade on target DNA containing C, 5-hmC or 5-ghmC (indicated by black arrow) at increasing protein concentrations [nM]. Fraction of bound target is indicated by white arrows, dotted lines represent separate gels.
    Figure Legend Snippet: Effect of T4 DNA modifications on type I-E CRISPR–Cas sgRNA mediated DNA targeting. ( A ) Schematic of DNA targeting and R-loop formation by Cascade. Modified cytosine residues are indicated in red. ( B ) Cleavage assay of Cas3 in conjunction with Cascade on 98 bp modified targets, indicated by black arrow. The marker is indicated by white arrows. Cascade effector complexes are loaded with either targeting crRNA (T crRNA) or non-targeting crRNA (NT crRNA). Restriction products of Cas3 are of undefined length. ( C ). Electrophoretic Mobility Shift Assay (EMSA) of Cascade on target DNA containing C, 5-hmC or 5-ghmC (indicated by black arrow) at increasing protein concentrations [nM]. Fraction of bound target is indicated by white arrows, dotted lines represent separate gels.

    Techniques Used: CRISPR, Modification, Cleavage Assay, Marker, Electrophoretic Mobility Shift Assay

    Effect of T4 DNA modifications on type V-A CRISPR–Cas sgRNA mediated DNA targeting. ( A ) Schematic of DNA targeting by Cas12a. Modified cytosine residues are indicated in red. Cleavage sites are indicated by black arrows. ( B ) Cleavage assay of Cas12a on 98 bp modified targets (indicated by black arrow). Cas12a is loaded with either targeting crRNA (C crRNA) or non-targeting crRNA (NC crRNA). Cleavage products of Cas12a are 49 and 44 bp. The marker is indicated by white arrows. ( C ) Electrophoretic Mobility Shift Assay (EMSA) of Cas12a on target DNA containing C, 5-hmC or 5-ghmC (indicated by black arrow) at increasing protein concentrations [nM]. Fraction of bound target is indicated by white arrows.
    Figure Legend Snippet: Effect of T4 DNA modifications on type V-A CRISPR–Cas sgRNA mediated DNA targeting. ( A ) Schematic of DNA targeting by Cas12a. Modified cytosine residues are indicated in red. Cleavage sites are indicated by black arrows. ( B ) Cleavage assay of Cas12a on 98 bp modified targets (indicated by black arrow). Cas12a is loaded with either targeting crRNA (C crRNA) or non-targeting crRNA (NC crRNA). Cleavage products of Cas12a are 49 and 44 bp. The marker is indicated by white arrows. ( C ) Electrophoretic Mobility Shift Assay (EMSA) of Cas12a on target DNA containing C, 5-hmC or 5-ghmC (indicated by black arrow) at increasing protein concentrations [nM]. Fraction of bound target is indicated by white arrows.

    Techniques Used: CRISPR, Modification, Cleavage Assay, Marker, Electrophoretic Mobility Shift Assay

    19) Product Images from "Quantitative measurement of transcriptional inhibition and mutagenesis induced by site-specifically incorporated DNA lesions in vitro and in vivo"

    Article Title: Quantitative measurement of transcriptional inhibition and mutagenesis induced by site-specifically incorporated DNA lesions in vitro and in vivo

    Journal: Nature protocols

    doi: 10.1038/nprot.2015.094

    The parent vector and competitor vector used in this study. ( a ) Plasmid maps of the parent vector (i.e., pTGFP-T7-Hha10T) and the competitor vector (i.e., pTGFP-T7-Hha10comp). ( b ) Sequences of the parent and competitor vectors between the NheI and EcoRI
    Figure Legend Snippet: The parent vector and competitor vector used in this study. ( a ) Plasmid maps of the parent vector (i.e., pTGFP-T7-Hha10T) and the competitor vector (i.e., pTGFP-T7-Hha10comp). ( b ) Sequences of the parent and competitor vectors between the NheI and EcoRI

    Techniques Used: Plasmid Preparation

    20) Product Images from "Identification of an hepatitis delta virus-like ribozyme at the mRNA 5?-end of the L1Tc retrotransposon from Trypanosoma cruzi"

    Article Title: Identification of an hepatitis delta virus-like ribozyme at the mRNA 5?-end of the L1Tc retrotransposon from Trypanosoma cruzi

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr478

    Analysis of the 5′-hydroxyl nature of the ends of the cleavage 3′-products. Schematic cleavage reaction of the clone pGEM-T easy −61/L1Tc+77 RNA is represented in ( A ). The uncleaved RNA is expected to have 5′-triphosphate and 3′-hydroxyl ends. The cleavage 5′- and 3′-products are expected to have 2′,3′-cyclic phosphate and 5′-hydroxyl ends, respectively. The T4 polynucleotide kinase (T4 PNK) challenge is represented in ( B ). 5′-hydroxyl ends, not 5′-phosphate, are sensible to phosphorylation by T4 PNK. Same quantity of endogenously radiolabeled cleavage fragments was both ice preserved in reaction buffer and phosphorylated by T4 PNK using gamma 32 P-ATP as phosphate donor. The cleavage 3′-products of clones 7134, 55 and pGEM-T easy RNAs were further radiolabeled confirming the expected 5′-hydroxyl nature of their 5′-ends (solid arrowhead). The 61 nt in length RNA 5′-product of the cleavage of the pGEM-T easy construct is used as negative control in the phosphorylation reaction (the empty arrow indicates the labeled 5′-product). One of the 3′-products is pre-treated with alkaline phosphatase prior to being treated with T4 PNK. (marked with an asterisk).
    Figure Legend Snippet: Analysis of the 5′-hydroxyl nature of the ends of the cleavage 3′-products. Schematic cleavage reaction of the clone pGEM-T easy −61/L1Tc+77 RNA is represented in ( A ). The uncleaved RNA is expected to have 5′-triphosphate and 3′-hydroxyl ends. The cleavage 5′- and 3′-products are expected to have 2′,3′-cyclic phosphate and 5′-hydroxyl ends, respectively. The T4 polynucleotide kinase (T4 PNK) challenge is represented in ( B ). 5′-hydroxyl ends, not 5′-phosphate, are sensible to phosphorylation by T4 PNK. Same quantity of endogenously radiolabeled cleavage fragments was both ice preserved in reaction buffer and phosphorylated by T4 PNK using gamma 32 P-ATP as phosphate donor. The cleavage 3′-products of clones 7134, 55 and pGEM-T easy RNAs were further radiolabeled confirming the expected 5′-hydroxyl nature of their 5′-ends (solid arrowhead). The 61 nt in length RNA 5′-product of the cleavage of the pGEM-T easy construct is used as negative control in the phosphorylation reaction (the empty arrow indicates the labeled 5′-product). One of the 3′-products is pre-treated with alkaline phosphatase prior to being treated with T4 PNK. (marked with an asterisk).

    Techniques Used: Clone Assay, Construct, Negative Control, Labeling

    21) 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

    22) 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

    23) Product Images from "FFPEcap-seq: a method for sequencing capped RNAs in formalin-fixed paraffin-embedded samples"

    Article Title: FFPEcap-seq: a method for sequencing capped RNAs in formalin-fixed paraffin-embedded samples

    Journal: Genome Research

    doi: 10.1101/gr.249656.119

    FFPEcap-seq overview. During enzymatic pretreatment, RNA is treated with T4 Polynucleotide Kinase (PNK) to phosphorylate 5′ hydroxyls and Terminator Nuclease to degrade uncapped RNAs. Sequence tags are added to the 5′ end of the cDNA during the reverse transcription reaction using template switching. cDNA is then amplified, and additional sequences are added during PCR amplification. (OH) Hydroxyl; (P) phosphate; (G r ) a guanine ribonucleotide; (N) an equal mix of nucleotides.
    Figure Legend Snippet: FFPEcap-seq overview. During enzymatic pretreatment, RNA is treated with T4 Polynucleotide Kinase (PNK) to phosphorylate 5′ hydroxyls and Terminator Nuclease to degrade uncapped RNAs. Sequence tags are added to the 5′ end of the cDNA during the reverse transcription reaction using template switching. cDNA is then amplified, and additional sequences are added during PCR amplification. (OH) Hydroxyl; (P) phosphate; (G r ) a guanine ribonucleotide; (N) an equal mix of nucleotides.

    Techniques Used: Sequencing, Amplification, Polymerase Chain Reaction

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

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

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

    doi: 10.1073/pnas.1603217113

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

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

    25) Product Images from "Telomerase abrogates aneuploidy-induced telomere replication stress, senescence and cell depletion"

    Article Title: Telomerase abrogates aneuploidy-induced telomere replication stress, senescence and cell depletion

    Journal: The EMBO Journal

    doi: 10.15252/embj.201490070

    Telomerase rescues aneuploidy-induced replication stress Early passages of BJ and BJ-hTERT fibroblasts were infected with aneuploidy-inducing shRNAs targeting the indicated genes or with a scrambled shRNA. A–C Analysis of phosphorylated RPA2 (pRPA2) foci by immunofluorescence staining: quantification of nuclear pRPA2 foci in (A) BJ and (B) BJ-hTERT fibroblasts; (C) representative images of pRPA2 foci (scale bar: 50 μm). Note that telomerase expression completely suppresses the induction of pRPA2 foci in BJ fibroblasts infected with aneuploidy-inducing shRNAs (B). D Co-staining of pRPA2 foci and telomeric DNA by FISH was used to quantify the number of pRPA2 foci co-localizing to telomeres in cells infected with aneuploidy-inducing shRNAs compared to scrambled shRNA-infected cells. E Representative image of immuno-FISH shows co-localization of pRPA2 foci and telomeric DNA. Here, 5 of 6 pRPA2 foci (dashed arrows) co-localize with telomeres (scale bar: 10 μm). F–H Chromatin immunoprecipitation (ChIP) was carried out using a pRPA2 antibody on lysates of cells infected with aneuploidy-inducing shRNAs or scrambled shRNA control. qPCR-based quantification of telomeric DNA in the immunoprecipitate of (F) BJ and (G) BJ-hTERT cells. (H) Quantification of telomeric DNA (telomere probe) and non-telomeric DNA (Alu probe) in the immunoprecipitate by dot blot and radioactive labeling. IgG-ChIP was used as a negative control. Note that aneuploidy-inducing shRNAs induced pRPA2 binding at telomeric DNA, which was rescued by telomerase expression. There was no detectable increase in pRPA2 foci at chromosomal localizations outside telomeres. I–L Quantification of cell cycle-dependent accumulation of pRPA2 foci formation in BJ cell infected with an aneuploidy-inducing shRNA against RXFP1 or with a scrambled shRNA control. Serum-starved, G1-arrested cells were re-stimulated and labeled at different time points with EdU. (I) Representative 2D cell cycle profile (EdU versus DAPI) at 24 h post-release from serum starvation. (J) Representative pictures of pRPA2 staining at the indicated cell cycle stages (scale bar: 10 μm). Quantification of pRPA2 foci at the indicated cell cycle stages in shRNA RXFP1- and scrambled shRNA-infected (K) BJ and (L) BJ-hTERT cells. Note the significant increase in pRPA2 foci in shRNA RXFP1-infected cells in late S-phase. Data information: (A, B, D) Mean ± SEM, two-tailed t -test, n ≥ 50 cells per sample. (F, G) Mean ± SEM, two-tailed t -test, n = 3 replicates. (K, L) Mean ± SEM, one-way ANOVA + Tukey’s test, n = 3–7 replicates. Source data are available online for this figure.
    Figure Legend Snippet: Telomerase rescues aneuploidy-induced replication stress Early passages of BJ and BJ-hTERT fibroblasts were infected with aneuploidy-inducing shRNAs targeting the indicated genes or with a scrambled shRNA. A–C Analysis of phosphorylated RPA2 (pRPA2) foci by immunofluorescence staining: quantification of nuclear pRPA2 foci in (A) BJ and (B) BJ-hTERT fibroblasts; (C) representative images of pRPA2 foci (scale bar: 50 μm). Note that telomerase expression completely suppresses the induction of pRPA2 foci in BJ fibroblasts infected with aneuploidy-inducing shRNAs (B). D Co-staining of pRPA2 foci and telomeric DNA by FISH was used to quantify the number of pRPA2 foci co-localizing to telomeres in cells infected with aneuploidy-inducing shRNAs compared to scrambled shRNA-infected cells. E Representative image of immuno-FISH shows co-localization of pRPA2 foci and telomeric DNA. Here, 5 of 6 pRPA2 foci (dashed arrows) co-localize with telomeres (scale bar: 10 μm). F–H Chromatin immunoprecipitation (ChIP) was carried out using a pRPA2 antibody on lysates of cells infected with aneuploidy-inducing shRNAs or scrambled shRNA control. qPCR-based quantification of telomeric DNA in the immunoprecipitate of (F) BJ and (G) BJ-hTERT cells. (H) Quantification of telomeric DNA (telomere probe) and non-telomeric DNA (Alu probe) in the immunoprecipitate by dot blot and radioactive labeling. IgG-ChIP was used as a negative control. Note that aneuploidy-inducing shRNAs induced pRPA2 binding at telomeric DNA, which was rescued by telomerase expression. There was no detectable increase in pRPA2 foci at chromosomal localizations outside telomeres. I–L Quantification of cell cycle-dependent accumulation of pRPA2 foci formation in BJ cell infected with an aneuploidy-inducing shRNA against RXFP1 or with a scrambled shRNA control. Serum-starved, G1-arrested cells were re-stimulated and labeled at different time points with EdU. (I) Representative 2D cell cycle profile (EdU versus DAPI) at 24 h post-release from serum starvation. (J) Representative pictures of pRPA2 staining at the indicated cell cycle stages (scale bar: 10 μm). Quantification of pRPA2 foci at the indicated cell cycle stages in shRNA RXFP1- and scrambled shRNA-infected (K) BJ and (L) BJ-hTERT cells. Note the significant increase in pRPA2 foci in shRNA RXFP1-infected cells in late S-phase. Data information: (A, B, D) Mean ± SEM, two-tailed t -test, n ≥ 50 cells per sample. (F, G) Mean ± SEM, two-tailed t -test, n = 3 replicates. (K, L) Mean ± SEM, one-way ANOVA + Tukey’s test, n = 3–7 replicates. Source data are available online for this figure.

    Techniques Used: Infection, shRNA, Immunofluorescence, Staining, Expressing, Fluorescence In Situ Hybridization, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Dot Blot, Labeling, Negative Control, Binding Assay, Two Tailed Test

    Aneuploidy induction in primary human fibroblasts provokes DNA damage responses (DDR) that are suppressed by telomerase Early passages of BJ and BJ-hTERT fibroblasts were infected with aneuploidy-inducing shRNAs or a scrambled shRNA. A–D Analysis of DNA damage foci. (A–C) Quantification of nuclear 53BP1 foci: comparison of scrambled shRNA versus aneuploidy-inducing shRNAs in (A) BJ and (B) BJ-hTERT fibroblasts, and (C) comparison of BJ versus BJ-hTERT fibroblasts transduced with aneuploidy-inducing shRNAs (pooled data from shRNAs against GJB3, RXFP1, OSBPL3, STARD9). (D) Representative images of 53BP1 foci (scale bar: 50 μm). E–H Metaphase spreads were stained with a telomere-specific probe. The number of multi-telomeric signals (MTS) was analyzed ( n = 15–30 metaphases per sample): comparison of scrambled shRNA versus aneuploidy-inducing shRNAs in (E) BJ and (F) BJ-hTERT fibroblasts. (G) Comparison of BJ versus BJ-hTERT fibroblasts transduced with aneuploidy-inducing shRNAs (pooled data from shRNAs against GJB3, RXFP1, OSBPL3, STARD9). (H) Representative images of metaphases derived from aneuploidy-induced BJ cells (scale bar: 5 μm). The arrows indicate multi-telomeric signals at the chromosome ends. I–M Quantification of BLM-coated anaphase bridges (APB). The percentage of Bloom coating was determined in those anaphases showing chromatid bridges ( n = 30–50 APBs per sample were analyzed from asynchronously growing cells): comparison of scrambled shRNA versus aneuploidy-inducing shRNAs in (I) BJ and (J) BJ-hTERT fibroblasts. (K) Comparison of BJ versus BJ-hTERT fibroblasts transduced with aneuploidy-inducing shRNAs (pooled data from shRNAs against RXFP1, OSBPL3). (L, M) Representative images of an anaphase bridge (L) and a BLM-coated anaphase bridge (M) (scale bar: 5 μm). Data information: All histograms depict mean values ± SEM; P -values were calculated by two-tailed t -test.
    Figure Legend Snippet: Aneuploidy induction in primary human fibroblasts provokes DNA damage responses (DDR) that are suppressed by telomerase Early passages of BJ and BJ-hTERT fibroblasts were infected with aneuploidy-inducing shRNAs or a scrambled shRNA. A–D Analysis of DNA damage foci. (A–C) Quantification of nuclear 53BP1 foci: comparison of scrambled shRNA versus aneuploidy-inducing shRNAs in (A) BJ and (B) BJ-hTERT fibroblasts, and (C) comparison of BJ versus BJ-hTERT fibroblasts transduced with aneuploidy-inducing shRNAs (pooled data from shRNAs against GJB3, RXFP1, OSBPL3, STARD9). (D) Representative images of 53BP1 foci (scale bar: 50 μm). E–H Metaphase spreads were stained with a telomere-specific probe. The number of multi-telomeric signals (MTS) was analyzed ( n = 15–30 metaphases per sample): comparison of scrambled shRNA versus aneuploidy-inducing shRNAs in (E) BJ and (F) BJ-hTERT fibroblasts. (G) Comparison of BJ versus BJ-hTERT fibroblasts transduced with aneuploidy-inducing shRNAs (pooled data from shRNAs against GJB3, RXFP1, OSBPL3, STARD9). (H) Representative images of metaphases derived from aneuploidy-induced BJ cells (scale bar: 5 μm). The arrows indicate multi-telomeric signals at the chromosome ends. I–M Quantification of BLM-coated anaphase bridges (APB). The percentage of Bloom coating was determined in those anaphases showing chromatid bridges ( n = 30–50 APBs per sample were analyzed from asynchronously growing cells): comparison of scrambled shRNA versus aneuploidy-inducing shRNAs in (I) BJ and (J) BJ-hTERT fibroblasts. (K) Comparison of BJ versus BJ-hTERT fibroblasts transduced with aneuploidy-inducing shRNAs (pooled data from shRNAs against RXFP1, OSBPL3). (L, M) Representative images of an anaphase bridge (L) and a BLM-coated anaphase bridge (M) (scale bar: 5 μm). Data information: All histograms depict mean values ± SEM; P -values were calculated by two-tailed t -test.

    Techniques Used: Infection, shRNA, Transduction, Staining, Derivative Assay, Two Tailed Test

    Model for aneuploidy-induced proliferation arrest and telomerase-induced survival of aneuploid cells Schematic diagram representing how aneuploidy induced by single-gene knockdown could lead to senescence by the induction of replication stress at telomeres and DNA damage. Telomerase activity alleviates telomere replication stress and facilitates continuous proliferation of aneuploidy cells.
    Figure Legend Snippet: Model for aneuploidy-induced proliferation arrest and telomerase-induced survival of aneuploid cells Schematic diagram representing how aneuploidy induced by single-gene knockdown could lead to senescence by the induction of replication stress at telomeres and DNA damage. Telomerase activity alleviates telomere replication stress and facilitates continuous proliferation of aneuploidy cells.

    Techniques Used: Activity Assay

    Impaired telomere replication in the absence of telomerase in mouse HSPCs A, B Knockdown of the indicated genes by mouse-specific shRNAs (shGJB3 and shOSBPL3) induces aneuploidy in mouse hematopoietic stem cells (HSCs). (A) Metaphase countings and (B) representative metaphase pictures from mouse lineage-negative HSPCs derived from the transplanted mice ( n : numbers are indicated). Fisher’s exact test was used to calculate significance. C In vivo survival of murine HSPCs (telomerase positive or negative) carrying shRNA (GFP-expressing vector) of control luciferase (left), Gjb3 (middle) and Osbpl3 (right), assessed from transplantation assay in lethally irradiated mice. See Materials and Methods for experimental details. Bar diagram shows 6-weeks GFP chimerism of CD45.1 (wild-type, WT) and CD45.2 (TERC −/− ) cells in peripheral blood of transplanted mice (mean ± SEM, two-tailed t -test, n = 15–16 mice/group). D, E Quantification of DNA replication patterns at telomeres in freshly isolated G1-TERC (D) or wild-type (E) HSPCs with the shOsbpl3 vector or, as a control, shLuciferase vector. See (F) for representative pictures. Counted DNA molecule numbers: n = 80 for G1-Luci, n = 99 for G1 Osbpl3, n = 100 for WT-Luci and n = 79 for WT-Osbpl3. Error bars indicate the standard error of the mean (SEM). P -values were calculated by chi-square test. F Representative images of DNA replication patterns occurring at telomeres. A fork arresting at a telomere was defined as an event in which the DNA replication signal stops within 2 kb from the telomeric border; partial telomere replication was defined as an event in which the DNA replication signal is only partially overlapping in length with the telomeric signal; complete telomere replication was defined as an event in which the DNA replication signal completely overlaps with the telomeric signal. Scale bar: 60 kb. 1: merge; 2: PNA-Telomeric Probe (red); 3: IdU (blue); 4: CldU (green). Freshly isolated wild-type (WT) or telomerase knockout (G1-TERC) mouse HSPCs carrying shRNA against Osbpl3 or, as a control, against firefly luciferase were labeled with thymidine analogues following a pulse and chase protocol. DNA was then isolated, combed and processed by immuno-FISH (see Supplementary Fig S7 for details).
    Figure Legend Snippet: Impaired telomere replication in the absence of telomerase in mouse HSPCs A, B Knockdown of the indicated genes by mouse-specific shRNAs (shGJB3 and shOSBPL3) induces aneuploidy in mouse hematopoietic stem cells (HSCs). (A) Metaphase countings and (B) representative metaphase pictures from mouse lineage-negative HSPCs derived from the transplanted mice ( n : numbers are indicated). Fisher’s exact test was used to calculate significance. C In vivo survival of murine HSPCs (telomerase positive or negative) carrying shRNA (GFP-expressing vector) of control luciferase (left), Gjb3 (middle) and Osbpl3 (right), assessed from transplantation assay in lethally irradiated mice. See Materials and Methods for experimental details. Bar diagram shows 6-weeks GFP chimerism of CD45.1 (wild-type, WT) and CD45.2 (TERC −/− ) cells in peripheral blood of transplanted mice (mean ± SEM, two-tailed t -test, n = 15–16 mice/group). D, E Quantification of DNA replication patterns at telomeres in freshly isolated G1-TERC (D) or wild-type (E) HSPCs with the shOsbpl3 vector or, as a control, shLuciferase vector. See (F) for representative pictures. Counted DNA molecule numbers: n = 80 for G1-Luci, n = 99 for G1 Osbpl3, n = 100 for WT-Luci and n = 79 for WT-Osbpl3. Error bars indicate the standard error of the mean (SEM). P -values were calculated by chi-square test. F Representative images of DNA replication patterns occurring at telomeres. A fork arresting at a telomere was defined as an event in which the DNA replication signal stops within 2 kb from the telomeric border; partial telomere replication was defined as an event in which the DNA replication signal is only partially overlapping in length with the telomeric signal; complete telomere replication was defined as an event in which the DNA replication signal completely overlaps with the telomeric signal. Scale bar: 60 kb. 1: merge; 2: PNA-Telomeric Probe (red); 3: IdU (blue); 4: CldU (green). Freshly isolated wild-type (WT) or telomerase knockout (G1-TERC) mouse HSPCs carrying shRNA against Osbpl3 or, as a control, against firefly luciferase were labeled with thymidine analogues following a pulse and chase protocol. DNA was then isolated, combed and processed by immuno-FISH (see Supplementary Fig S7 for details).

    Techniques Used: Derivative Assay, Mouse Assay, In Vivo, shRNA, Expressing, Plasmid Preparation, Luciferase, Transplantation Assay, Irradiation, Two Tailed Test, Isolation, Knock-Out, Labeling, Fluorescence In Situ Hybridization

    26) Product Images from "Towards the Generation of an ASFV-pA104R DISC Mutant and a Complementary Cell Line—A Potential Methodology for the Production of a Vaccine Candidate"

    Article Title: Towards the Generation of an ASFV-pA104R DISC Mutant and a Complementary Cell Line—A Potential Methodology for the Production of a Vaccine Candidate

    Journal: Vaccines

    doi: 10.3390/vaccines7030068

    Schematic overview to generate helper cell lines expressing pA104R. Vero/COS-1 cells were transfected with the linearized pIRESneo_ASFV-A104R vector and then were subjected to antibiotic selective pressure. The surviving clones were expanded and the Vero-pA104R/COS-1-pA104R cell lines were established.
    Figure Legend Snippet: Schematic overview to generate helper cell lines expressing pA104R. Vero/COS-1 cells were transfected with the linearized pIRESneo_ASFV-A104R vector and then were subjected to antibiotic selective pressure. The surviving clones were expanded and the Vero-pA104R/COS-1-pA104R cell lines were established.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Clone Assay

    Open reading frame (ORF) A104R was successfully integrated in transfected Vero and COS-1 cells. ORF A104R was amplified by PCR (287 bp) from the genome of Vero-pA104R clones, cultured under antibiotic selective pressure (G418). Line 1: Molecular marker (NZYDNA Ladder III); Line 2: Vero wild type (wt, negative control); Line 3: pIRESneo A104R (positive control); Lines 4–6: three distinct Vero-pA104R clones, transfected with linearized plasmid and Lipofectamine 2000; Lines 7,8: two distinct Vero-pA104R clones, transfected with circular plasmid and Lipofectamine 2000; Line 9: COS-1 wt cells (negative control); Line 10: COS-1 cells transfected by Lipofectamine 2000 with the A104R construct; Line 11: Flp-In wt cells; Line 12: Flp-In cells transformed with the A104R construct; Lines 13–15: COS-1 cells transfected with Lipofectamine LTX (16 weeks); Line 16: FRT/A104R plasmid (positive control); Lines 17–20: Flp-In clones sustaining the selective pressure.
    Figure Legend Snippet: Open reading frame (ORF) A104R was successfully integrated in transfected Vero and COS-1 cells. ORF A104R was amplified by PCR (287 bp) from the genome of Vero-pA104R clones, cultured under antibiotic selective pressure (G418). Line 1: Molecular marker (NZYDNA Ladder III); Line 2: Vero wild type (wt, negative control); Line 3: pIRESneo A104R (positive control); Lines 4–6: three distinct Vero-pA104R clones, transfected with linearized plasmid and Lipofectamine 2000; Lines 7,8: two distinct Vero-pA104R clones, transfected with circular plasmid and Lipofectamine 2000; Line 9: COS-1 wt cells (negative control); Line 10: COS-1 cells transfected by Lipofectamine 2000 with the A104R construct; Line 11: Flp-In wt cells; Line 12: Flp-In cells transformed with the A104R construct; Lines 13–15: COS-1 cells transfected with Lipofectamine LTX (16 weeks); Line 16: FRT/A104R plasmid (positive control); Lines 17–20: Flp-In clones sustaining the selective pressure.

    Techniques Used: Transfection, Amplification, Polymerase Chain Reaction, Clone Assay, Cell Culture, Marker, Negative Control, Positive Control, Plasmid Preparation, Construct, Transformation Assay

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

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

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-14-888

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

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

    28) 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

    29) 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

    30) Product Images from "Endosulfan upregulates AP-1 binding and ARE-mediated transcription via ERK1/2 and p38 activation in HepG2 cells"

    Article Title: Endosulfan upregulates AP-1 binding and ARE-mediated transcription via ERK1/2 and p38 activation in HepG2 cells

    Journal: Toxicology

    doi: 10.1016/j.tox.2011.11.013

    Effect of endosulfan on AP-1 binding. AP-1 binding was measured by EMSA with 5′- 32 P-end-labeled double-stranded oligonucleotides after 24-h exposure to endosulfan (Endo). AP-1 probe was end-labeled with T4 polynucleotide kinase. For competition
    Figure Legend Snippet: Effect of endosulfan on AP-1 binding. AP-1 binding was measured by EMSA with 5′- 32 P-end-labeled double-stranded oligonucleotides after 24-h exposure to endosulfan (Endo). AP-1 probe was end-labeled with T4 polynucleotide kinase. For competition

    Techniques Used: Binding Assay, Labeling

    31) Product Images from "Antha-guided Automation of Darwin Assembly for the Construction of Bespoke Gene Libraries"

    Article Title: Antha-guided Automation of Darwin Assembly for the Construction of Bespoke Gene Libraries

    Journal: bioRxiv

    doi: 10.1101/2019.12.17.879486

    Darwin Assembly with biotinylated oligonucleotides. Schematic representation of Darwin Assembly with biotinylated oligonucleotides (adapted from Cozens and Pinheiro, 2017 [ 2 ]). Using a single recognition site in the plasmid, a nicking endonuclease and exonuclease III are added to degrade one strand of the template. A 5’-boundary oligonucleotide containing a 5’-biotinTEG modification (shown as a blue dot), a 3’-boundary oligonucleotide containing a protected 3’-end (3’-3’dT, not shown) and inner oligonucleotides are annealed onto the single stranded template. The Darwin Assembly master mix including a polymerase, ligase and dNTPs is used to extend and ligate the primers, creating the library.The assembled construct is captured via biotin-streptavidin pulldown using paramagnetic beads (shown in brown). The boundary oligonucleotides also contain PCR priming sites and Type IIs restriction sites (shown as reds overhangs) which are used for PCR recovery and subcloning into the expression backbone. The protocol is fully automated up to isothermal assembly and we are now optimizing the automation for capture, clean-up and PCR recovery steps.
    Figure Legend Snippet: Darwin Assembly with biotinylated oligonucleotides. Schematic representation of Darwin Assembly with biotinylated oligonucleotides (adapted from Cozens and Pinheiro, 2017 [ 2 ]). Using a single recognition site in the plasmid, a nicking endonuclease and exonuclease III are added to degrade one strand of the template. A 5’-boundary oligonucleotide containing a 5’-biotinTEG modification (shown as a blue dot), a 3’-boundary oligonucleotide containing a protected 3’-end (3’-3’dT, not shown) and inner oligonucleotides are annealed onto the single stranded template. The Darwin Assembly master mix including a polymerase, ligase and dNTPs is used to extend and ligate the primers, creating the library.The assembled construct is captured via biotin-streptavidin pulldown using paramagnetic beads (shown in brown). The boundary oligonucleotides also contain PCR priming sites and Type IIs restriction sites (shown as reds overhangs) which are used for PCR recovery and subcloning into the expression backbone. The protocol is fully automated up to isothermal assembly and we are now optimizing the automation for capture, clean-up and PCR recovery steps.

    Techniques Used: Plasmid Preparation, Modification, Construct, Polymerase Chain Reaction, Subcloning, Expressing

    32) 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

    33) Product Images from "Mapping the Human miRNA Interactome by CLASH Reveals Frequent Noncanonical Binding"

    Article Title: Mapping the Human miRNA Interactome by CLASH Reveals Frequent Noncanonical Binding

    Journal: Cell

    doi: 10.1016/j.cell.2013.03.043

    Experimental Validation of CLASH Identified miR-92a Targets, Related to Figure 5 Changes in mRNA abundance upon miR-92a depletion in PTH-AGO1-HEK293 cells, measured by Affymetrix microarrays. The performance of various classes of miR-92a targets identified in CLASH analyses, and targets containing the miR-92a motif, are compared to transcripts containing a match to the miR-92a 7-mer seed sequence (positive control), to random transcripts, and to targets lacking a match to the miR-92a 7-mer seed (negative control). The left and right edge of the box represent 25th and 75th percentile, respectively. The ends of the whiskers show the minimum and maximum values of the data.
    Figure Legend Snippet: Experimental Validation of CLASH Identified miR-92a Targets, Related to Figure 5 Changes in mRNA abundance upon miR-92a depletion in PTH-AGO1-HEK293 cells, measured by Affymetrix microarrays. The performance of various classes of miR-92a targets identified in CLASH analyses, and targets containing the miR-92a motif, are compared to transcripts containing a match to the miR-92a 7-mer seed sequence (positive control), to random transcripts, and to targets lacking a match to the miR-92a 7-mer seed (negative control). The left and right edge of the box represent 25th and 75th percentile, respectively. The ends of the whiskers show the minimum and maximum values of the data.

    Techniques Used: Sequencing, Positive Control, Negative Control

    Overview of Experimental and Bioinformatic Procedures (A) Growing cells were UV irradiated, and PTH-AGO1 was purified. RNA fragmentation, ligation, cDNA synthesis, and sequencing of AGO1-associated RNAs allowed the identification of sites of AGO1 binding (as single reads) and RNA-RNA interactions at AGO1-binding sites (as chimeric reads). (B) Sequencing reads were mapped to a database of human transcripts using BLAST ( Altschul et al., 1990 ). Sequences reliably mapped to two different sites were folded in silico using UNAFold ( Markham and Zuker, 2008 ) to identify the interaction site of the RNA molecules that gave rise to the chimeric cDNA. (C) Example interaction between miR-196a/b and HOXC8 that was supported by chimeric reads (red), and a cluster of nonchimeric reads (green). The blue dashed line represents the location of the miRNA bit of chimera, and the red dashed line shows the 25 nt mRNA extension added during the analysis. The interaction was previously shown experimentally ( Li et al., 2010 ) and can be predicted by RNAhybrid ( Rehmsmeier et al., 2004 ). (D) Distribution of all miRNA interactions among various classes of RNAs. The main miRNA targets are mRNAs and are represented by 18,514 interactions. See also Figure S1 and Tables S1 and S2 A–S2C.
    Figure Legend Snippet: Overview of Experimental and Bioinformatic Procedures (A) Growing cells were UV irradiated, and PTH-AGO1 was purified. RNA fragmentation, ligation, cDNA synthesis, and sequencing of AGO1-associated RNAs allowed the identification of sites of AGO1 binding (as single reads) and RNA-RNA interactions at AGO1-binding sites (as chimeric reads). (B) Sequencing reads were mapped to a database of human transcripts using BLAST ( Altschul et al., 1990 ). Sequences reliably mapped to two different sites were folded in silico using UNAFold ( Markham and Zuker, 2008 ) to identify the interaction site of the RNA molecules that gave rise to the chimeric cDNA. (C) Example interaction between miR-196a/b and HOXC8 that was supported by chimeric reads (red), and a cluster of nonchimeric reads (green). The blue dashed line represents the location of the miRNA bit of chimera, and the red dashed line shows the 25 nt mRNA extension added during the analysis. The interaction was previously shown experimentally ( Li et al., 2010 ) and can be predicted by RNAhybrid ( Rehmsmeier et al., 2004 ). (D) Distribution of all miRNA interactions among various classes of RNAs. The main miRNA targets are mRNAs and are represented by 18,514 interactions. See also Figure S1 and Tables S1 and S2 A–S2C.

    Techniques Used: Irradiation, Purification, Ligation, Sequencing, Binding Assay, In Silico

    34) Product Images from "Bacteriophage DNA glucosylation impairs target DNA binding by type I and II but not by type V CRISPR–Cas effector complexes"

    Article Title: Bacteriophage DNA glucosylation impairs target DNA binding by type I and II but not by type V CRISPR–Cas effector complexes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1264

    Effect of T4 DNA modifications on type II-A CRISPR–Cas sgRNA mediated DNA targeting. ( A ) Schematic of DNA targeting by Cas9. Modified cytosine residues are indicated in red. Cleavage sites are indicated by black arrows. ( B ) Cleavage assay of Cas9 on 98 bp modified targets (indicated by black arrow). Cas9 is loaded with either targeting sgRNA (T sgRNA) or non-targeting sgRNA (NT sgRNA). Restriction products of Cas9 are 61 and 37 bp. ( C ) EMSA of dCas9 on target DNA containing C, 5-hmC or 5-ghmC (indicated by black arrow) at increasing protein concentrations [nM]. Fraction of bound target is indicated by white arrows.
    Figure Legend Snippet: Effect of T4 DNA modifications on type II-A CRISPR–Cas sgRNA mediated DNA targeting. ( A ) Schematic of DNA targeting by Cas9. Modified cytosine residues are indicated in red. Cleavage sites are indicated by black arrows. ( B ) Cleavage assay of Cas9 on 98 bp modified targets (indicated by black arrow). Cas9 is loaded with either targeting sgRNA (T sgRNA) or non-targeting sgRNA (NT sgRNA). Restriction products of Cas9 are 61 and 37 bp. ( C ) EMSA of dCas9 on target DNA containing C, 5-hmC or 5-ghmC (indicated by black arrow) at increasing protein concentrations [nM]. Fraction of bound target is indicated by white arrows.

    Techniques Used: CRISPR, Modification, Cleavage Assay

    Effect of T4 DNA modifications on type I-E CRISPR–Cas sgRNA mediated DNA targeting. ( A ) Schematic of DNA targeting and R-loop formation by Cascade. Modified cytosine residues are indicated in red. ( B ) Cleavage assay of Cas3 in conjunction with Cascade on 98 bp modified targets, indicated by black arrow. The marker is indicated by white arrows. Cascade effector complexes are loaded with either targeting crRNA (T crRNA) or non-targeting crRNA (NT crRNA). Restriction products of Cas3 are of undefined length. ( C ). Electrophoretic Mobility Shift Assay (EMSA) of Cascade on target DNA containing C, 5-hmC or 5-ghmC (indicated by black arrow) at increasing protein concentrations [nM]. Fraction of bound target is indicated by white arrows, dotted lines represent separate gels.
    Figure Legend Snippet: Effect of T4 DNA modifications on type I-E CRISPR–Cas sgRNA mediated DNA targeting. ( A ) Schematic of DNA targeting and R-loop formation by Cascade. Modified cytosine residues are indicated in red. ( B ) Cleavage assay of Cas3 in conjunction with Cascade on 98 bp modified targets, indicated by black arrow. The marker is indicated by white arrows. Cascade effector complexes are loaded with either targeting crRNA (T crRNA) or non-targeting crRNA (NT crRNA). Restriction products of Cas3 are of undefined length. ( C ). Electrophoretic Mobility Shift Assay (EMSA) of Cascade on target DNA containing C, 5-hmC or 5-ghmC (indicated by black arrow) at increasing protein concentrations [nM]. Fraction of bound target is indicated by white arrows, dotted lines represent separate gels.

    Techniques Used: CRISPR, Modification, Cleavage Assay, Marker, Electrophoretic Mobility Shift Assay

    Potential steric clashes between target nucleotide 5-ghmC modifications and CRISPR effector proteins. ( A ) Multiple clashes (indicated in red) are observed between the polypeptide chains of Cascade and 5-ghmC modifications of nucleotides in the target strand (TS, complementary to the crRNA) and non-target strand (NTS). ( B ) Clashes are mostly observed between the polypeptide chains of Cas9 and 5-ghmC modifications of nucleotides in the seed region. ( C ) No clashes are observed in the seed region for Cas12a.
    Figure Legend Snippet: Potential steric clashes between target nucleotide 5-ghmC modifications and CRISPR effector proteins. ( A ) Multiple clashes (indicated in red) are observed between the polypeptide chains of Cascade and 5-ghmC modifications of nucleotides in the target strand (TS, complementary to the crRNA) and non-target strand (NTS). ( B ) Clashes are mostly observed between the polypeptide chains of Cas9 and 5-ghmC modifications of nucleotides in the seed region. ( C ) No clashes are observed in the seed region for Cas12a.

    Techniques Used: CRISPR

    Effect of T4 DNA modifications on type V-A CRISPR–Cas sgRNA mediated DNA targeting. ( A ) Schematic of DNA targeting by Cas12a. Modified cytosine residues are indicated in red. Cleavage sites are indicated by black arrows. ( B ) Cleavage assay of Cas12a on 98 bp modified targets (indicated by black arrow). Cas12a is loaded with either targeting crRNA (C crRNA) or non-targeting crRNA (NC crRNA). Cleavage products of Cas12a are 49 and 44 bp. The marker is indicated by white arrows. ( C ) Electrophoretic Mobility Shift Assay (EMSA) of Cas12a on target DNA containing C, 5-hmC or 5-ghmC (indicated by black arrow) at increasing protein concentrations [nM]. Fraction of bound target is indicated by white arrows.
    Figure Legend Snippet: Effect of T4 DNA modifications on type V-A CRISPR–Cas sgRNA mediated DNA targeting. ( A ) Schematic of DNA targeting by Cas12a. Modified cytosine residues are indicated in red. Cleavage sites are indicated by black arrows. ( B ) Cleavage assay of Cas12a on 98 bp modified targets (indicated by black arrow). Cas12a is loaded with either targeting crRNA (C crRNA) or non-targeting crRNA (NC crRNA). Cleavage products of Cas12a are 49 and 44 bp. The marker is indicated by white arrows. ( C ) Electrophoretic Mobility Shift Assay (EMSA) of Cas12a on target DNA containing C, 5-hmC or 5-ghmC (indicated by black arrow) at increasing protein concentrations [nM]. Fraction of bound target is indicated by white arrows.

    Techniques Used: CRISPR, Modification, Cleavage Assay, Marker, Electrophoretic Mobility Shift Assay

    35) Product Images from "Comparative analysis of Cas6b processing and CRISPR RNA stability"

    Article Title: Comparative analysis of Cas6b processing and CRISPR RNA stability

    Journal: RNA Biology

    doi: 10.4161/rna.23715

    Figure 4. In-line crRNA probing assays. ( A ) Individual spacer(n)-repeat-spacer(n+1) RNAs were generated by in vitro run-off transcription, processed by Cas6b to yield mature crRNAs. The crRNAs were 5′ labeled with (γ- 32 P)-ATP and
    Figure Legend Snippet: Figure 4. In-line crRNA probing assays. ( A ) Individual spacer(n)-repeat-spacer(n+1) RNAs were generated by in vitro run-off transcription, processed by Cas6b to yield mature crRNAs. The crRNAs were 5′ labeled with (γ- 32 P)-ATP and

    Techniques Used: Generated, In Vitro, Labeling

    36) Product Images from "RNA binding protein FXR1-miR301a-3p axis contributes to p21WAF1 degradation in oral cancer"

    Article Title: RNA binding protein FXR1-miR301a-3p axis contributes to p21WAF1 degradation in oral cancer

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1008580

    PNPT1 is overexpressed in HNSCC that degrades miR301a-3p in the absence of FXR1. ( A ) qRT-PCR analysis of FXR1 , XRN1 , XRN2 , and PNPT1 in HNSCC cell lines compared to primary line HOK along with lung cancer cell line A549. Both ACTIN and RPS18 served as endogenous controls. ( B ) (up) qRT-PCR analyses to test the expression of miR301a-3p in UMSCC74B cells under individual and double knockdown of FXR1 and PNPT1. RNU6 served as an endogenous control. (down) Northern hybridization of total RNA isolated from UMSCC74B cells under individual and double knockdown of FXR1 and PNPT1 shows the levels of miR301a-3p. RNU6 is used as an endogenous control. 5S rRNA is used as a loading control. ( C ) qRT-PCR analysis of FXR1 , p21 , and PNPT1 in UMSCC74B cells under individual and double knockdown of FXR1 and PNPT1. Both ACTIN and RPS18 served as endogenous controls. ( D ) Western blot analysis of FXR1, p21, and PNPT1 in UMSCC74B under individual and double knockdown of FXR1 and PNPT1. GAPDH serves as an endogenous control. ( E ) EMSA with 5’-labeled miR301a-3p with rec. FXR1 and rec. PNPT1 proteins. L1: RNA only, L2: RNA + FXR1, L3: RNA + PNPT1, L4: RNA + FXR1 (5 pmole) for 15 min, followed by PNPT1 (5 pmole) for another 15 min. L5-7: samples are loaded 2 hours after the first loading of from L1,3, and 4 to visualize the bottom of the gel. Statistical significance ( p -value): *
    Figure Legend Snippet: PNPT1 is overexpressed in HNSCC that degrades miR301a-3p in the absence of FXR1. ( A ) qRT-PCR analysis of FXR1 , XRN1 , XRN2 , and PNPT1 in HNSCC cell lines compared to primary line HOK along with lung cancer cell line A549. Both ACTIN and RPS18 served as endogenous controls. ( B ) (up) qRT-PCR analyses to test the expression of miR301a-3p in UMSCC74B cells under individual and double knockdown of FXR1 and PNPT1. RNU6 served as an endogenous control. (down) Northern hybridization of total RNA isolated from UMSCC74B cells under individual and double knockdown of FXR1 and PNPT1 shows the levels of miR301a-3p. RNU6 is used as an endogenous control. 5S rRNA is used as a loading control. ( C ) qRT-PCR analysis of FXR1 , p21 , and PNPT1 in UMSCC74B cells under individual and double knockdown of FXR1 and PNPT1. Both ACTIN and RPS18 served as endogenous controls. ( D ) Western blot analysis of FXR1, p21, and PNPT1 in UMSCC74B under individual and double knockdown of FXR1 and PNPT1. GAPDH serves as an endogenous control. ( E ) EMSA with 5’-labeled miR301a-3p with rec. FXR1 and rec. PNPT1 proteins. L1: RNA only, L2: RNA + FXR1, L3: RNA + PNPT1, L4: RNA + FXR1 (5 pmole) for 15 min, followed by PNPT1 (5 pmole) for another 15 min. L5-7: samples are loaded 2 hours after the first loading of from L1,3, and 4 to visualize the bottom of the gel. Statistical significance ( p -value): *

    Techniques Used: Quantitative RT-PCR, Expressing, Northern Blot, Hybridization, Isolation, Western Blot, Labeling

    FXR1 and miR301a-3p cooperatively target and repress p21 expression. Model representation of FXR1 mediated stabilization of miR301a-3p from exonuclease PNPT1 to regulate p21 translation in cancer cells. Left: In cancer cells, FXR1 binds and protects mature miR301a-3p from exonuclease mediated degradation. FXR1-miRNA complex binds and degrades p21 3’-UTR. Right: In FXR1 depleted cancer cells, mature miR301a-3p is produced and degraded by 3’-5’ exonuclease PNPT1. As a result, p21 is stabilized in the cells resulting in a p21 protein upregulation.
    Figure Legend Snippet: FXR1 and miR301a-3p cooperatively target and repress p21 expression. Model representation of FXR1 mediated stabilization of miR301a-3p from exonuclease PNPT1 to regulate p21 translation in cancer cells. Left: In cancer cells, FXR1 binds and protects mature miR301a-3p from exonuclease mediated degradation. FXR1-miRNA complex binds and degrades p21 3’-UTR. Right: In FXR1 depleted cancer cells, mature miR301a-3p is produced and degraded by 3’-5’ exonuclease PNPT1. As a result, p21 is stabilized in the cells resulting in a p21 protein upregulation.

    Techniques Used: Expressing, Produced

    FXR1 and miR301a-3p cooperatively target and repress p21 expression. ( A ) qRT-PCR analyses of miR301a-3p in UMSCC74B cells treated with miRNA mimic and scrambled control in the presence and absence of FXR1. RNU6 serves as an endogenous control. ( B ) qRT-PCR analyses of FXR1 and p21 in UMSCC74B cells treated with miRNA mimic and a scrambled control in the presence and absence of FXR1. ACTIN and RPS18 serve as an endogenous control. ( C ) Western blot analyses of FXR1 and p21 from UMSCC74B treated with miRNA mimic and a scrambled control in the presence and absence of FXR1. β-Actin serves as a loading control. ( D ) qRT-PCR analyses of FXR1 and p21 in UMSCC74B cells transfected with p21 overexpression plasmid and vector control. ACTIN and RPS18 serve as an endogenous control. ( E ) Western blot analyses of FXR1 and p21 from UMSCC74B cells transfected with p21 overexpression plasmid and vector control. β-Actin serves as a loading control. ( F ) qRT-PCR analyses of miR301a-3p in UMSCC74B cells transfected with p21 overexpression plasmid and vector control. RNU6 serves as an endogenous control. ( G ) Tumor image from 12 mice injected with UMSCC74B cells expressing IPTG inducible control and FXR1 shRNA clones and treated with 10mM IPTG/5% glucose in the drinking water. Tumors obtained from Male# 4–6 were used for western blot analyses for FXR1 and p21 where β-Actin serves as a loading control and qRT-PCR for miR301a-3p, RNU6 serves as an endogenous control. Data here represent the mean of n = 3 experiments. Statistical significance ( p -value): *
    Figure Legend Snippet: FXR1 and miR301a-3p cooperatively target and repress p21 expression. ( A ) qRT-PCR analyses of miR301a-3p in UMSCC74B cells treated with miRNA mimic and scrambled control in the presence and absence of FXR1. RNU6 serves as an endogenous control. ( B ) qRT-PCR analyses of FXR1 and p21 in UMSCC74B cells treated with miRNA mimic and a scrambled control in the presence and absence of FXR1. ACTIN and RPS18 serve as an endogenous control. ( C ) Western blot analyses of FXR1 and p21 from UMSCC74B treated with miRNA mimic and a scrambled control in the presence and absence of FXR1. β-Actin serves as a loading control. ( D ) qRT-PCR analyses of FXR1 and p21 in UMSCC74B cells transfected with p21 overexpression plasmid and vector control. ACTIN and RPS18 serve as an endogenous control. ( E ) Western blot analyses of FXR1 and p21 from UMSCC74B cells transfected with p21 overexpression plasmid and vector control. β-Actin serves as a loading control. ( F ) qRT-PCR analyses of miR301a-3p in UMSCC74B cells transfected with p21 overexpression plasmid and vector control. RNU6 serves as an endogenous control. ( G ) Tumor image from 12 mice injected with UMSCC74B cells expressing IPTG inducible control and FXR1 shRNA clones and treated with 10mM IPTG/5% glucose in the drinking water. Tumors obtained from Male# 4–6 were used for western blot analyses for FXR1 and p21 where β-Actin serves as a loading control and qRT-PCR for miR301a-3p, RNU6 serves as an endogenous control. Data here represent the mean of n = 3 experiments. Statistical significance ( p -value): *

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Over Expression, Plasmid Preparation, Mouse Assay, Injection, shRNA, Clone Assay

    miR301a-3p targets p21 mRNA to repress translation. ( A ) A schematic showing miR301a-3p binding sites on p21 3’UTR. Gq signifies the G-quadruplex regions on p21 3’-UTR for FXR1 binding as well. Gq regions on human p21 3’-UTR is segregated into high Gq (red) and low Gq (pink) bearing regions. miR301a-3p has a binding site in each of the high and low Gq region. Binding sites are shown in the figure with miRNA base-pairing. Mutant bases (to disrupt the miRNA-mRNA duplex) are shown in red. ( B ) qRT-PCR analyses of miR301a-3p in UMSCC74B cells treated with miRNA inhibitor and a scrambled control. RNU6 serves as an endogenous control. ( C ) p21 protein is up-regulated in miR301a-3p inhibitor transfected UMSCC74B cells. β-Actin serves as a loading control. ( D ) p21 3’UTR luciferase activity is significantly up-regulated in the presence of miR301a-3p inhibitor in UMSCC74B cells compared to the scrambled control transfected cells. Cells were collected forty-eight hours post- transfection with miRNA control and 301a-3p inhibitor along with empty 3’UTR luciferase plasmid and wild type p21 3′UTR, the lysates were analyzed for luciferase activity using a luminometer. The empty 3’UTR luciferase plasmid served as a transfection and loading control. Values here are the means ± SD from three independent experiments by using an unpaired two-sample t-test. ( E ) Expression of miR301a-3p in UMSCC74B cells treated with miRNA mimics compared to scrambled control. RNU6 served as an endogenous control. ( F ) p21 protein is down-regulated in miR301a-3p mimic treated cells compared to control. β-Actin serves as a loading control. ( G ) p21 3’UTR luciferase activity is significantly down-regulated in the miR301a-3p mimic treated UMSCC74B cells compared to the scrambled mimic treated cells. However, the luciferase activity in mut1 and mut2 is seg1 and the seg2 region of miR301a-3p binding does not change after mimic transfection. Experiments were performed as described in (D). Statistical significance ( p -value): *
    Figure Legend Snippet: miR301a-3p targets p21 mRNA to repress translation. ( A ) A schematic showing miR301a-3p binding sites on p21 3’UTR. Gq signifies the G-quadruplex regions on p21 3’-UTR for FXR1 binding as well. Gq regions on human p21 3’-UTR is segregated into high Gq (red) and low Gq (pink) bearing regions. miR301a-3p has a binding site in each of the high and low Gq region. Binding sites are shown in the figure with miRNA base-pairing. Mutant bases (to disrupt the miRNA-mRNA duplex) are shown in red. ( B ) qRT-PCR analyses of miR301a-3p in UMSCC74B cells treated with miRNA inhibitor and a scrambled control. RNU6 serves as an endogenous control. ( C ) p21 protein is up-regulated in miR301a-3p inhibitor transfected UMSCC74B cells. β-Actin serves as a loading control. ( D ) p21 3’UTR luciferase activity is significantly up-regulated in the presence of miR301a-3p inhibitor in UMSCC74B cells compared to the scrambled control transfected cells. Cells were collected forty-eight hours post- transfection with miRNA control and 301a-3p inhibitor along with empty 3’UTR luciferase plasmid and wild type p21 3′UTR, the lysates were analyzed for luciferase activity using a luminometer. The empty 3’UTR luciferase plasmid served as a transfection and loading control. Values here are the means ± SD from three independent experiments by using an unpaired two-sample t-test. ( E ) Expression of miR301a-3p in UMSCC74B cells treated with miRNA mimics compared to scrambled control. RNU6 served as an endogenous control. ( F ) p21 protein is down-regulated in miR301a-3p mimic treated cells compared to control. β-Actin serves as a loading control. ( G ) p21 3’UTR luciferase activity is significantly down-regulated in the miR301a-3p mimic treated UMSCC74B cells compared to the scrambled mimic treated cells. However, the luciferase activity in mut1 and mut2 is seg1 and the seg2 region of miR301a-3p binding does not change after mimic transfection. Experiments were performed as described in (D). Statistical significance ( p -value): *

    Techniques Used: Binding Assay, Mutagenesis, Quantitative RT-PCR, Transfection, Luciferase, Activity Assay, Plasmid Preparation, Expressing

    FXR1 modulates the expression of a subset of miRNAs in oral cancer cells. ( A ) Volcano plot of differential miRNA expression in UMSCC74B cells in the absence of FXR1. Red: up-regulated and green: down-regulated miRNAs in the absence of FXR1. The inset shows the knockdown efficiency of FXR1 by shRNA (TRCN0000159153) compared to a scrambled shRNA where GAPDH serves as a loading control. X-axis signifies the log 2 values of the miRNA fold change where y-axis plots the -log 10 - p -values of the differentially expressed miRNAs in the FXR1 knockdown cells compared to control. ( B ) qRT-PCR of the altered miRNAs in FXR1 knockdown UMSCC74B cells. RNU6 serves as an endogenous control. Inset shows the knockdown efficiency of FXR1 by shRNA (TRCN0000159153) compared to a scrambled shRNA where β-Actin serves as a loading control. ( C ) qRT-PCR is showing that miR301a-3p is significantly down-regulated in all the FXR1 knockdown HNSCC cell lines. RNU6 serves as an endogenous control. ( D ) qRT-PCR is showing that premiR301a is not altered in most of the FXR1 knockdown HNSCC cell lines except Cal27. 18S rRNA serves as an endogenous control. Data from B-D represent the mean of n = 3 experiments. Statistical significance ( p -value): *
    Figure Legend Snippet: FXR1 modulates the expression of a subset of miRNAs in oral cancer cells. ( A ) Volcano plot of differential miRNA expression in UMSCC74B cells in the absence of FXR1. Red: up-regulated and green: down-regulated miRNAs in the absence of FXR1. The inset shows the knockdown efficiency of FXR1 by shRNA (TRCN0000159153) compared to a scrambled shRNA where GAPDH serves as a loading control. X-axis signifies the log 2 values of the miRNA fold change where y-axis plots the -log 10 - p -values of the differentially expressed miRNAs in the FXR1 knockdown cells compared to control. ( B ) qRT-PCR of the altered miRNAs in FXR1 knockdown UMSCC74B cells. RNU6 serves as an endogenous control. Inset shows the knockdown efficiency of FXR1 by shRNA (TRCN0000159153) compared to a scrambled shRNA where β-Actin serves as a loading control. ( C ) qRT-PCR is showing that miR301a-3p is significantly down-regulated in all the FXR1 knockdown HNSCC cell lines. RNU6 serves as an endogenous control. ( D ) qRT-PCR is showing that premiR301a is not altered in most of the FXR1 knockdown HNSCC cell lines except Cal27. 18S rRNA serves as an endogenous control. Data from B-D represent the mean of n = 3 experiments. Statistical significance ( p -value): *

    Techniques Used: Expressing, shRNA, Quantitative RT-PCR

    The stability of miR301a-3p is FXR1 dependent. ( A ) qRT-PCR assay of FXR1 knockdown UMSCC74B cells showing significant down- and up-regulation of FXR1 and p21 , respectively, compared to control. AGO2 did not show any change after the FXR1 knockdown. Both ACTIN and RPS18 served as endogenous controls. ( B ) Western blot analyses of FXR1, p21, and AGO2 from UMSCC74B cells collected at different time points as mentioned after shFXR1 transduction. GAPDH serves as a loading control. ( C ) qRT-PCR assay of FXR1 knockdown UMSCC74B cells showing significant miR301a-3p decay from 48hrs compared to control. Cells were collected at the designated time points after shRNA transduction. RNU6 serves as an endogenous control. ( D ) UMSCC74B cells stably expressing IPTG inducible shControl and shFXR1 were treated with 1mM IPTG for 72 hrs, followed by actinomycin D (ActD) or DMSO treatment for 8 hrs. Total RNA was prepared from all samples for northern hybridization. RNU6 serves as an endogenous control whereas total sample loading was shown by the 5s rRNA level from the PAGE. The same samples were used for western blot to show the knockdown of FXR1. Statistical significance ( p -value): *
    Figure Legend Snippet: The stability of miR301a-3p is FXR1 dependent. ( A ) qRT-PCR assay of FXR1 knockdown UMSCC74B cells showing significant down- and up-regulation of FXR1 and p21 , respectively, compared to control. AGO2 did not show any change after the FXR1 knockdown. Both ACTIN and RPS18 served as endogenous controls. ( B ) Western blot analyses of FXR1, p21, and AGO2 from UMSCC74B cells collected at different time points as mentioned after shFXR1 transduction. GAPDH serves as a loading control. ( C ) qRT-PCR assay of FXR1 knockdown UMSCC74B cells showing significant miR301a-3p decay from 48hrs compared to control. Cells were collected at the designated time points after shRNA transduction. RNU6 serves as an endogenous control. ( D ) UMSCC74B cells stably expressing IPTG inducible shControl and shFXR1 were treated with 1mM IPTG for 72 hrs, followed by actinomycin D (ActD) or DMSO treatment for 8 hrs. Total RNA was prepared from all samples for northern hybridization. RNU6 serves as an endogenous control whereas total sample loading was shown by the 5s rRNA level from the PAGE. The same samples were used for western blot to show the knockdown of FXR1. Statistical significance ( p -value): *

    Techniques Used: Quantitative RT-PCR, Western Blot, Transduction, shRNA, Stable Transfection, Expressing, Northern Blot, Hybridization, Polyacrylamide Gel Electrophoresis

    FXR1 binds to miR301a-3p in vivo and in vitro. ( A ) RNA-immunoprecipitation shows miRNA301a-3p binds to FXR1 in UMSCC74B cells compared to control mouse IgG. Both ACTIN and RPS18 served as endogenous controls. FXR1 antibody pull-down efficiency by immunoprecipitation is shown in the inset. ( B ) SDS-PAGE showing rec. FXR1 purification from the Ni-NTA column. ( C ) EMSA with 5’-labeled miR301a-3p and rec. FXR1 protein. 0.5 pmole of [y- 32 P] ATP labeled miRNA was mock-treated or mixed with increasing concentration of recombinant FXR1 protein and incubated at 37°C for 30 min. Free RNA and miRNP complexes are shown in the figure.
    Figure Legend Snippet: FXR1 binds to miR301a-3p in vivo and in vitro. ( A ) RNA-immunoprecipitation shows miRNA301a-3p binds to FXR1 in UMSCC74B cells compared to control mouse IgG. Both ACTIN and RPS18 served as endogenous controls. FXR1 antibody pull-down efficiency by immunoprecipitation is shown in the inset. ( B ) SDS-PAGE showing rec. FXR1 purification from the Ni-NTA column. ( C ) EMSA with 5’-labeled miR301a-3p and rec. FXR1 protein. 0.5 pmole of [y- 32 P] ATP labeled miRNA was mock-treated or mixed with increasing concentration of recombinant FXR1 protein and incubated at 37°C for 30 min. Free RNA and miRNP complexes are shown in the figure.

    Techniques Used: In Vivo, In Vitro, Immunoprecipitation, SDS Page, Purification, Labeling, Concentration Assay, Recombinant, Incubation

    37) Product Images from "Genomic Characterization of Sixteen Yersinia enterocolitica-Infecting Podoviruses of Pig Origin"

    Article Title: Genomic Characterization of Sixteen Yersinia enterocolitica-Infecting Podoviruses of Pig Origin

    Journal: Viruses

    doi: 10.3390/v10040174

    The genomic map of fPS-7. The predicted genes are arranged in the direction of transcription shown by different colored arrows. Genes involved in nucleotide metabolism, DNA replication, recombination or repair are shown in green. Genes involved in morphogenesis and virion structures are depicted in brown. Genes involved in DNA packaging and lysis, are shown in blue and red, respectively. Genes coding for hypothetical proteins or conserved phage proteins of unknown function are shown in light grey. Homing endonucleases are shown in yellow. Direct terminal repeats (DTRs) are shown in black. On top of the genome, the host RNA polymerase (RNAP)-dependent promoters are shown with red double-arrows labelled with −35 and −10, and the phage RNAP-dependent promoters with black arrows labelled from P1 to P12. Terminators are shown along the genome as purple triangles and labelled from T1 to T4. The genetic map was created using the Geneious software.
    Figure Legend Snippet: The genomic map of fPS-7. The predicted genes are arranged in the direction of transcription shown by different colored arrows. Genes involved in nucleotide metabolism, DNA replication, recombination or repair are shown in green. Genes involved in morphogenesis and virion structures are depicted in brown. Genes involved in DNA packaging and lysis, are shown in blue and red, respectively. Genes coding for hypothetical proteins or conserved phage proteins of unknown function are shown in light grey. Homing endonucleases are shown in yellow. Direct terminal repeats (DTRs) are shown in black. On top of the genome, the host RNA polymerase (RNAP)-dependent promoters are shown with red double-arrows labelled with −35 and −10, and the phage RNAP-dependent promoters with black arrows labelled from P1 to P12. Terminators are shown along the genome as purple triangles and labelled from T1 to T4. The genetic map was created using the Geneious software.

    Techniques Used: Lysis, Software

    38) Product Images from "Impaired Translesion Synthesis in Xeroderma Pigmentosum Variant Extracts"

    Article Title: Impaired Translesion Synthesis in Xeroderma Pigmentosum Variant Extracts

    Journal: Molecular and Cellular Biology

    doi:

    TLS past and AAF adduct by extracts from normal or XPV cells. Analysis of TLS catalyzed by 30 μg of either normal (N; 205BR) or XPV (XP6DU) cell extracts. DNA products obtained after 1 h of incubation with pUC-3G1.ss or pUC-3G3.ss were cleaved with enzymes Pvu II and Eco RI and subjected to electrophoresis on an 10% polyacrylamide–7 M urea denaturing gel. L−1, L0, and L+1 are products generated if synthesis is blocked one nucleotide before, opposite, and one nucleotide after the lesion, respectively. TLS0 and TLS−1 are products from TLS via nonslipped and slipped intermediates. As an internal standard, an identical amount of a 120-nucleotide fragment (end labeled with [γ- 32 P]ATP by T4 polynucleotide kinase) was added to each reaction at the end of the replication step.
    Figure Legend Snippet: TLS past and AAF adduct by extracts from normal or XPV cells. Analysis of TLS catalyzed by 30 μg of either normal (N; 205BR) or XPV (XP6DU) cell extracts. DNA products obtained after 1 h of incubation with pUC-3G1.ss or pUC-3G3.ss were cleaved with enzymes Pvu II and Eco RI and subjected to electrophoresis on an 10% polyacrylamide–7 M urea denaturing gel. L−1, L0, and L+1 are products generated if synthesis is blocked one nucleotide before, opposite, and one nucleotide after the lesion, respectively. TLS0 and TLS−1 are products from TLS via nonslipped and slipped intermediates. As an internal standard, an identical amount of a 120-nucleotide fragment (end labeled with [γ- 32 P]ATP by T4 polynucleotide kinase) was added to each reaction at the end of the replication step.

    Techniques Used: Incubation, Electrophoresis, Generated, Labeling

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

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

    Journal: JCI Insight

    doi: 10.1172/jci.insight.127317

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

    Techniques Used:

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

    Techniques Used: RNA Sequencing Assay, Isolation, Purification

    40) Product Images from "A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA"

    Article Title: A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr051

    The β-gt can specifically modify 5hmC residues at a high efficiency. ( a ) Oligonucleotides that were either incubated in the presence or absence of the β-gt were digested with Taq I, treated with alkaline phosphatase, 5′-end labeled using T4 polynucleotide kinase and digested to 5′-mononucleotides using DNase I and Snake Venom Phosphodiesterase. Radiolabeled mononucleotides were analyzed by two-dimensional TLC. C, 3′-deoxyribocytosine-5′-monophosphate; T, 3′-deoxyribothymidine-5′-monophosphate; 5meC, 3′-deoxyribo-N5-methylcytosine-5′-monophosphate; 5hmC, 3′-deoxyribo-N5-hydroxymethylcytosine-5′-monophosphate. ( b ) HPLC coupled to tandem mass spectrometry was used to measure the efficiency of the β-gt reaction. Substrates analyzed were 2.7 kb linear PCR products of pUC18: the dC substrate contained only cytosine residues; the 5meC substrate was created by methylating the CpG dinucleotide of the cytosine substrate; the 5hmC substrate was created by using d5hmC in place of dCTP in the PCR reactions; the β-glu-5hmC substrate was created by incubating the 5hmC substrate with the β-gt in the presence of UDP-glucose. Control DNA was prepared from salmon sperm. LC/MS/MS chromatograms of the cytosine residues from each of the substrates are presented. Abbreviations: dC, 3′-deoxyribocytosine; 5me(dC), 3′-deoxyribo-N5-methylcytosine; 5hm(dC), 3′-deoxyribo-N5-hydroxymethylcytosine; 5-glu-hm(dC), 3′-deoxyribo-N5-(β- d -glucosyl(hydroxymethyl))cytosine. Asterisks indictes that cytosines are only 5meC modified at CpG sequences.
    Figure Legend Snippet: The β-gt can specifically modify 5hmC residues at a high efficiency. ( a ) Oligonucleotides that were either incubated in the presence or absence of the β-gt were digested with Taq I, treated with alkaline phosphatase, 5′-end labeled using T4 polynucleotide kinase and digested to 5′-mononucleotides using DNase I and Snake Venom Phosphodiesterase. Radiolabeled mononucleotides were analyzed by two-dimensional TLC. C, 3′-deoxyribocytosine-5′-monophosphate; T, 3′-deoxyribothymidine-5′-monophosphate; 5meC, 3′-deoxyribo-N5-methylcytosine-5′-monophosphate; 5hmC, 3′-deoxyribo-N5-hydroxymethylcytosine-5′-monophosphate. ( b ) HPLC coupled to tandem mass spectrometry was used to measure the efficiency of the β-gt reaction. Substrates analyzed were 2.7 kb linear PCR products of pUC18: the dC substrate contained only cytosine residues; the 5meC substrate was created by methylating the CpG dinucleotide of the cytosine substrate; the 5hmC substrate was created by using d5hmC in place of dCTP in the PCR reactions; the β-glu-5hmC substrate was created by incubating the 5hmC substrate with the β-gt in the presence of UDP-glucose. Control DNA was prepared from salmon sperm. LC/MS/MS chromatograms of the cytosine residues from each of the substrates are presented. Abbreviations: dC, 3′-deoxyribocytosine; 5me(dC), 3′-deoxyribo-N5-methylcytosine; 5hm(dC), 3′-deoxyribo-N5-hydroxymethylcytosine; 5-glu-hm(dC), 3′-deoxyribo-N5-(β- d -glucosyl(hydroxymethyl))cytosine. Asterisks indictes that cytosines are only 5meC modified at CpG sequences.

    Techniques Used: Incubation, Labeling, Thin Layer Chromatography, High Performance Liquid Chromatography, Mass Spectrometry, Polymerase Chain Reaction, Liquid Chromatography with Mass Spectroscopy, Modification

    Related Articles

    Ligation:

    Article Title: High-throughput determination of RNA structure by proximity ligation
    Article Snippet: .. Following end-repair, complexes were immediately transferred to 450 uL ligation reaction mix (50 uL 10X T4 DNA Ligase Buffer w/ 10 mM ATP (NEB); 5 uL SuperASE-In (Ambion), 12.5 uL T4 RNA Ligase I (NEB), 382.5 uL 1X PBS w/ 0.2% IGEPAL), and incubated overnight in a 16 °C water bath, after which complexes were added to 1.5 mL TriZOL (Ambion). .. Samples were then purified using Direct-ZOL spin columns (Zymo) according to manufacturer's protocols.

    Labeling:

    Article Title: Nop5p Is a Small Nucleolar Ribonucleoprotein Component Required for Pre-18 S rRNA Processing in Yeast *
    Article Snippet: .. RNAs were 3′-end labeled with RNA ligase (New England Biolabs) using a standard method , purified, and electrophoresed on a 6% denaturing polyacrylamide gel. .. The “total” labeling sample mixture consisted of a portion of the supernatant fraction from the control IP treated, extracted, precipitated, and labeled as described above.

    Article Title: Biochemical and Biophysical Properties of a Putative Hub Protein Expressed by Vaccinia Virus *
    Article Snippet: .. This RNA (and RNA not subjected to the cleavage reaction) was then labeled in a reaction containing 150 μCi of [5′-32 P]pCp (3000 Ci/mmol; PerkinElmer Life Sciences) and 10 units of RNA ligase (New England Biolabs) at 4 °C overnight. .. The labeled RNA was then purified using phenol-chloroform extraction and ethanol purification.

    Purification:

    Article Title: Nop5p Is a Small Nucleolar Ribonucleoprotein Component Required for Pre-18 S rRNA Processing in Yeast *
    Article Snippet: .. RNAs were 3′-end labeled with RNA ligase (New England Biolabs) using a standard method , purified, and electrophoresed on a 6% denaturing polyacrylamide gel. .. The “total” labeling sample mixture consisted of a portion of the supernatant fraction from the control IP treated, extracted, precipitated, and labeled as described above.

    Sequencing:

    Article Title: A Leaderless Genome Identified during Persistent Bovine Coronavirus Infection Is Associated with Attenuation of Gene Expression
    Article Snippet: .. To determine the terminal sequence of viral negative-strand genomic RNA and sgmRNA, total cellular RNA was treated with tobacco acid pyrophosphatase (Epicentre), ligated with T4 RNA ligase I (New England Biolabs) and primer 1: BCV3′UTR1(−) was used for RT; for PCR, primers BCV3′UTR(−) and BCV107(+), and primers BCV3′UTR(−) and RYN(+) were used for determining terminal sequence of negative-strand genomic RNA and subgenomic mRNA, respectively. .. The resulting 50-µl PCR mixture was heated to 94°C for 2 min and subjected to 50 cycles of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C.

    Incubation:

    Article Title: A Leaderless Genome Identified during Persistent Bovine Coronavirus Infection Is Associated with Attenuation of Gene Expression
    Article Snippet: .. A 3-µl aliquot of 10X ligase buffer and 2 U (in 2 µl) of T4 RNA ligase I (New England Biolabs) were added, and the mixture was incubated for 16 h at 16°C. .. After ligation, RNA was phenol-chloroform-extracted and quantitated, and 1 µg of ligated RNA was used for an RT reaction to synthesize cDNA with SuperScript III reverse transcriptase (Invitrogen).

    Article Title: High-throughput determination of RNA structure by proximity ligation
    Article Snippet: .. Following end-repair, complexes were immediately transferred to 450 uL ligation reaction mix (50 uL 10X T4 DNA Ligase Buffer w/ 10 mM ATP (NEB); 5 uL SuperASE-In (Ambion), 12.5 uL T4 RNA Ligase I (NEB), 382.5 uL 1X PBS w/ 0.2% IGEPAL), and incubated overnight in a 16 °C water bath, after which complexes were added to 1.5 mL TriZOL (Ambion). .. Samples were then purified using Direct-ZOL spin columns (Zymo) according to manufacturer's protocols.

    Polymerase Chain Reaction:

    Article Title: A Leaderless Genome Identified during Persistent Bovine Coronavirus Infection Is Associated with Attenuation of Gene Expression
    Article Snippet: .. To determine the terminal sequence of viral negative-strand genomic RNA and sgmRNA, total cellular RNA was treated with tobacco acid pyrophosphatase (Epicentre), ligated with T4 RNA ligase I (New England Biolabs) and primer 1: BCV3′UTR1(−) was used for RT; for PCR, primers BCV3′UTR(−) and BCV107(+), and primers BCV3′UTR(−) and RYN(+) were used for determining terminal sequence of negative-strand genomic RNA and subgenomic mRNA, respectively. .. The resulting 50-µl PCR mixture was heated to 94°C for 2 min and subjected to 50 cycles of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C.

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    New England Biolabs t4 pnk buffer
    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 <t>T4</t> 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.
    T4 Pnk Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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.

    Journal: Molecular Therapy. Nucleic Acids

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

    doi: 10.1016/j.omtn.2017.07.008

    Figure Lengend 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.

    Article Snippet: The protocol was modified as follows when CIP or T4 PNK treatment is necessary: (1) for CIP treatment, in 20 μL of products from one in vitro transcription reaction before DNase treatment, we added 1 μL of DNase (supplied with T7 Transcription Kit), 1 μL of CIP, 4 μL of 10× CutSmart buffer (NEB), and water to total volume of 40 μL, and incubated at 37°C for 15 min; and (2) for T4 PNK treatment, in 20 μL of products from one in vitro transcription reaction before DNase treatment, we added 1 μL of DNase (supplied with T7 Transcription Kit), 1 μL of T4 PNK, 4 μL of 10× T4 PNK buffer (NEB), and water to total volume of 40 μL, and incubated at 37°C for 15 min. All T7 in vitro transcription products were purified by Micro Bio-Spin P-30 Gel Columns, Tris Buffer, from Bio-Rad.

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

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

    Journal: JCI Insight

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

    doi: 10.1172/jci.insight.127317

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

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

    Techniques:

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

    Journal: JCI Insight

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

    doi: 10.1172/jci.insight.127317

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

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

    Techniques: RNA Sequencing Assay, Isolation, Purification

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

    Journal: bioRxiv

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

    doi: 10.1101/465633

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

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

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

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-019-13991-9

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

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

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