rna  (New England Biolabs)


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    Name:
    NEBNext Ultra RNA Library Prep Kit for Illumina
    Description:
    NEBNext Ultra RNA Library Prep Kit for Illumina 96 rxns
    Catalog Number:
    E7530L
    Price:
    3494
    Category:
    mRNA Template Preparation for PCR
    Size:
    96 rxns
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    Structured Review

    New England Biolabs rna
    NEBNext Ultra RNA Library Prep Kit for Illumina
    NEBNext Ultra RNA Library Prep Kit for Illumina 96 rxns
    https://www.bioz.com/result/rna/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rna - by Bioz Stars, 2021-06
    86/100 stars

    Images

    1) Product Images from "Imprinting of the human L3MBTL gene, a polycomb family member located in a region of chromosome 20 deleted in human myeloid malignancies"

    Article Title: Imprinting of the human L3MBTL gene, a polycomb family member located in a region of chromosome 20 deleted in human myeloid malignancies

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

    doi: 10.1073/pnas.0308195101

    Methylation of CpG islands 3 and 4 is associated with transcriptional silencing. ( A ) Diagram of 5′ region of L3MBTL locus showing the Msc I site, PCR primers (P7, P8) and PCR products derived from either RNA or genomic DNA. * , A/G SNP in exon 2. ( B ) PCR amplification of genomic DNA or RNA from individual 1 was followed by Msc I digestion. Similar results were obtained in two independent experiments. Lane 1, undigested PCR product of granulocyte genomic DNA; lanes 2 and 3, Msc I digested PCR products of genomic DNA from granulocytes and T cells, respectively; lane 4, undigested RT-PCR product from T cells; lane 5, Msc I digested RT-PCR product from T cells.
    Figure Legend Snippet: Methylation of CpG islands 3 and 4 is associated with transcriptional silencing. ( A ) Diagram of 5′ region of L3MBTL locus showing the Msc I site, PCR primers (P7, P8) and PCR products derived from either RNA or genomic DNA. * , A/G SNP in exon 2. ( B ) PCR amplification of genomic DNA or RNA from individual 1 was followed by Msc I digestion. Similar results were obtained in two independent experiments. Lane 1, undigested PCR product of granulocyte genomic DNA; lanes 2 and 3, Msc I digested PCR products of genomic DNA from granulocytes and T cells, respectively; lane 4, undigested RT-PCR product from T cells; lane 5, Msc I digested RT-PCR product from T cells.

    Techniques Used: Methylation, Polymerase Chain Reaction, Derivative Assay, Amplification, Reverse Transcription Polymerase Chain Reaction

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1049

    DNA sequence of the RT-PCR products of 155 bp ( A ), 305 bp ( B ) and 188 bp ( C ) obtained by 3′5′ RACE from 16S rRNA m , p1 and p2 , respectively. The DNA sequences shown here are those found in the 16S ribosomal RNA. Sequences derived from the m 16S rRNA are highlighted in a grey background. Arrows indicate that complete DNA sequences extend until to the sequence of oligonucleotide FA, and to the reverse complement of oligonucleotide RA. In each panel, the two nucleotides shown in bold and underlined correspond to the junction, generated by the RNA ligation, of the 5′ and 3′ ends of the 16S RNAs.
    Figure Legend Snippet: DNA sequence of the RT-PCR products of 155 bp ( A ), 305 bp ( B ) and 188 bp ( C ) obtained by 3′5′ RACE from 16S rRNA m , p1 and p2 , respectively. The DNA sequences shown here are those found in the 16S ribosomal RNA. Sequences derived from the m 16S rRNA are highlighted in a grey background. Arrows indicate that complete DNA sequences extend until to the sequence of oligonucleotide FA, and to the reverse complement of oligonucleotide RA. In each panel, the two nucleotides shown in bold and underlined correspond to the junction, generated by the RNA ligation, of the 5′ and 3′ ends of the 16S RNAs.

    Techniques Used: Sequencing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Generated, Ligation

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

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

    ( A and B ) Sedimentation profiles of ribosomal subunits prepared from strains MC4100 pWKS30 (A) and MC4100 pDM39 (B) labelled with [ 3 H]-uridine at 45°C. Sedimentation is from right to left. A 260 /ml, open circles. [ 3 H] c.p.m. × 10 3 , filled circles. ( C ) Total RNAs phenol-extracted from the same two bacterial batches were subjected to 3′5′ RACE analysis without prior thermodenaturation of RNA (lane a = MC4100 pWSK30, lane b = MC4100 pDM39). Relative DNA concentrations in the bands of the agarose gel were estimated using a Typhoon Trio phosphorImager and the ImageQuant software.
    Figure Legend Snippet: ( A and B ) Sedimentation profiles of ribosomal subunits prepared from strains MC4100 pWKS30 (A) and MC4100 pDM39 (B) labelled with [ 3 H]-uridine at 45°C. Sedimentation is from right to left. A 260 /ml, open circles. [ 3 H] c.p.m. × 10 3 , filled circles. ( C ) Total RNAs phenol-extracted from the same two bacterial batches were subjected to 3′5′ RACE analysis without prior thermodenaturation of RNA (lane a = MC4100 pWSK30, lane b = MC4100 pDM39). Relative DNA concentrations in the bands of the agarose gel were estimated using a Typhoon Trio phosphorImager and the ImageQuant software.

    Techniques Used: Sedimentation, Agarose Gel Electrophoresis, Software

    ( A ) Preparative sucrose gradient sedimentation of ribosomal particles from strain MC4100 labelled with [ 3 H]-uridine at 45°C. Fractions 18–21 were pooled, and 21S particles were concentrated by ultracentrifugation. ( B ) An aliquot of the 21S particles isolated from ( A ) was mixed with unlabelled 50S and 30S subunits from a wt strain and rerun on a new sucrose gradient under the same conditions. Sedimentation is from right to left. A 260 /ml, open circles. [ 3 H] c.p.m. × 10 3 , filled circles. ( C ) 16S rRNA phenol-extracted from the 21S particles isolated from ( A ) were subjected to 3′5′ RACE analysis, with (lane b) or without (lane a) a thermodenaturation step prior to the 3′5′ RNA ligation. The RT-PCR products of 305 bp and 188 bp were purified by preparative agarose gel electrophoresis (from lanes a and b, respectively) and sequenced. In lane c, an aliquot of the 16S rRNA was thermodenatured, but instead of rapid freezing leading to irreversible RNA denaturation (as shown in lane b), was then subjected to a slow cooling (a couple of hours at room temperature) leading to reversible RNA denaturation, prior to the 3′5′ RACE procedure: the RT-PCR products obtained under these conditions are similar to those obtained in the absence of any RNA denaturation step (lane a).
    Figure Legend Snippet: ( A ) Preparative sucrose gradient sedimentation of ribosomal particles from strain MC4100 labelled with [ 3 H]-uridine at 45°C. Fractions 18–21 were pooled, and 21S particles were concentrated by ultracentrifugation. ( B ) An aliquot of the 21S particles isolated from ( A ) was mixed with unlabelled 50S and 30S subunits from a wt strain and rerun on a new sucrose gradient under the same conditions. Sedimentation is from right to left. A 260 /ml, open circles. [ 3 H] c.p.m. × 10 3 , filled circles. ( C ) 16S rRNA phenol-extracted from the 21S particles isolated from ( A ) were subjected to 3′5′ RACE analysis, with (lane b) or without (lane a) a thermodenaturation step prior to the 3′5′ RNA ligation. The RT-PCR products of 305 bp and 188 bp were purified by preparative agarose gel electrophoresis (from lanes a and b, respectively) and sequenced. In lane c, an aliquot of the 16S rRNA was thermodenatured, but instead of rapid freezing leading to irreversible RNA denaturation (as shown in lane b), was then subjected to a slow cooling (a couple of hours at room temperature) leading to reversible RNA denaturation, prior to the 3′5′ RACE procedure: the RT-PCR products obtained under these conditions are similar to those obtained in the absence of any RNA denaturation step (lane a).

    Techniques Used: Sedimentation, Isolation, Ligation, Reverse Transcription Polymerase Chain Reaction, Purification, Agarose Gel Electrophoresis

    3) Product Images from "Microgravity validation of a novel system for RNA isolation and multiplex quantitative real time PCR analysis of gene expression on the International Space Station"

    Article Title: Microgravity validation of a novel system for RNA isolation and multiplex quantitative real time PCR analysis of gene expression on the International Space Station

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0183480

    RNA isolation and RT-qPCR in microgravity. (A) Photo of SPM. (B) Typical RNA quality from SPM with E . coli (left panel) and mouse liver (right panel), control Qiagen (left lane) SPM (right lane). Center panel shows RNA quality from the 1 g control (left lane) and the returned microgravity sample from ISS (right lane). (C-E) Scatter plots with jitter of the microgravity and 1 g control E . coli singleplex (C), duplex (D) and triplex (E) reactions. One outlier is indicated by the open marker in C. One of the microgravity triplex tubes did not give a dnaK-FAM signal (E). (F-H) Scatter plots with jitter of the microgravity and 1 g control mouse liver singleplex (F), duplex (G) and triplex (H) reactions. One outlier from the microgravity triplex fn1 plot is indicated by the open marker and no gapdh-FAM signal was seen in the microgravity triplex reactions (H).
    Figure Legend Snippet: RNA isolation and RT-qPCR in microgravity. (A) Photo of SPM. (B) Typical RNA quality from SPM with E . coli (left panel) and mouse liver (right panel), control Qiagen (left lane) SPM (right lane). Center panel shows RNA quality from the 1 g control (left lane) and the returned microgravity sample from ISS (right lane). (C-E) Scatter plots with jitter of the microgravity and 1 g control E . coli singleplex (C), duplex (D) and triplex (E) reactions. One outlier is indicated by the open marker in C. One of the microgravity triplex tubes did not give a dnaK-FAM signal (E). (F-H) Scatter plots with jitter of the microgravity and 1 g control mouse liver singleplex (F), duplex (G) and triplex (H) reactions. One outlier from the microgravity triplex fn1 plot is indicated by the open marker and no gapdh-FAM signal was seen in the microgravity triplex reactions (H).

    Techniques Used: Isolation, Quantitative RT-PCR, Marker

    4) Product Images from "Global repositioning of transcription start sites in a plant-fermenting bacterium"

    Article Title: Global repositioning of transcription start sites in a plant-fermenting bacterium

    Journal: Nature Communications

    doi: 10.1038/ncomms13783

    Overview of the Capp-Switch sequencing approach. Capp-Switch includes ( a – c ) capture of 5′ mRNA fragments and ( d – f ) cDNA synthesis and sequencing. ( a ) The mRNA 5′ triphosphate is capped with biotin-GTP by VCE. ( b ) RNA is fragmented and ( c ) the capped 5′ mRNA fragments are captured on streptavidin magnetic beads and separated from other RNA. ( d ) The 5′ mRNA fragments are reverse transcribed to single-stranded cDNA using MMLV reverse transcriptase. An oligonucleotide hybridizes to the 3′ overhang and the complementary sequence is synthesized by the MMLV template-switching activity. ( e ) Double-stranded cDNA is synthesized using primers that hybridize to the single-stranded cDNA termini. ( f ) The cDNA is sequenced on a high-throughput platform.
    Figure Legend Snippet: Overview of the Capp-Switch sequencing approach. Capp-Switch includes ( a – c ) capture of 5′ mRNA fragments and ( d – f ) cDNA synthesis and sequencing. ( a ) The mRNA 5′ triphosphate is capped with biotin-GTP by VCE. ( b ) RNA is fragmented and ( c ) the capped 5′ mRNA fragments are captured on streptavidin magnetic beads and separated from other RNA. ( d ) The 5′ mRNA fragments are reverse transcribed to single-stranded cDNA using MMLV reverse transcriptase. An oligonucleotide hybridizes to the 3′ overhang and the complementary sequence is synthesized by the MMLV template-switching activity. ( e ) Double-stranded cDNA is synthesized using primers that hybridize to the single-stranded cDNA termini. ( f ) The cDNA is sequenced on a high-throughput platform.

    Techniques Used: Sequencing, Magnetic Beads, Synthesized, Activity Assay, High Throughput Screening Assay

    5) Product Images from "The cohesin regulator Stag1 promotes cell plasticity through heterochromatin regulation"

    Article Title: The cohesin regulator Stag1 promotes cell plasticity through heterochromatin regulation

    Journal: bioRxiv

    doi: 10.1101/2021.02.14.429938

    rRNA expression and nascent translation upon Stag1 loss. (A) Confocal images of IF to Nucleolin (Ncl) and Nascent RNA in ES cells treated with the siRNA panel. Nuclei were counterstained with DAPI. Note the accumulation of nascent RNA within the nucleolus. (B) Imaris quantification from (A) of the mean intensity of nascent RNA (EU-488) within the nucleoli, as defined by Nucleolin signal. Box plots and statistical analysis were done as before. Data are from two biological replicates, n > 50/condition, except for siSA1 5p where n > 35. ** p
    Figure Legend Snippet: rRNA expression and nascent translation upon Stag1 loss. (A) Confocal images of IF to Nucleolin (Ncl) and Nascent RNA in ES cells treated with the siRNA panel. Nuclei were counterstained with DAPI. Note the accumulation of nascent RNA within the nucleolus. (B) Imaris quantification from (A) of the mean intensity of nascent RNA (EU-488) within the nucleoli, as defined by Nucleolin signal. Box plots and statistical analysis were done as before. Data are from two biological replicates, n > 50/condition, except for siSA1 5p where n > 35. ** p

    Techniques Used: Expressing

    6) Product Images from "Aberrant adhesion impacts early development in a Dictyostelium model for juvenile neuronal ceroid lipofuscinosis"

    Article Title: Aberrant adhesion impacts early development in a Dictyostelium model for juvenile neuronal ceroid lipofuscinosis

    Journal: Cell Adhesion & Migration

    doi: 10.1080/19336918.2016.1236179

    RNA extraction, cDNA synthesis, and qPCR
    Figure Legend Snippet: RNA extraction, cDNA synthesis, and qPCR

    Techniques Used: RNA Extraction, Real-time Polymerase Chain Reaction

    7) Product Images from "Potato spindle tuber viroid infection triggers degradation of chloride channel protein CLC-b-like and Ribosomal protein S3a-like mRNAs in tomato plants"

    Article Title: Potato spindle tuber viroid infection triggers degradation of chloride channel protein CLC-b-like and Ribosomal protein S3a-like mRNAs in tomato plants

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-08823-z

    Validation of the predicted vd-sRNA:target mRNA complex formation by an artificial microRNA. ( A ) Duplexes predicted to be formed by complexes of amiRNAs and GFP reporter constructs containing the Chloride Channel CLC-b-like and the RPS3a-like mRNA target sequences. ( B ) N. benthamiana leaves were agro-infiltrated with (1) empty pBIN61 vector (EV) plus GFP:CLCb; (2) amiR:(+)191 plus GFP:CLCb; (3) EV plus GFP:RPS3a; and, (4) amiR:(−)69 plus GFP:RPS3a. At 3-dpi, the leaves were photographed under UV illumination. N. benthamiana leaves were agroinfiltrated in the same combinations as in ( B ). ( C ) At 3-dpi, total RNA extracts were subjected to RNA gel blot analyses with either GFP (top panel) or 7SL (lower panel) radiolabeled probes. Full size gel blots are presented in Fig. S2 . The signals in ( C ) were quantified and expressed as a ratio of the GFP to the 7SL signals. For each set of experiments, the ratio of GFP to 7SL obtained with EV plus GFP:XX (control) was set at a value of 1. The additional bars indicate the relative GFP/ 7SL ratio for each amiRNA (as indicated) expressed with its respective GFP:XX. ( D ) At 3 dpi, total protein extracts were subjected to immunoblotting with anti- GFP (top panel) and anti-PEPC (lower panel) antibodies. Full size immunoblots are presented in Fig. S3 . The immunoblot signals from ( D ) were quantified and expressed as a ratio of the GFP to the PEPC signals. For each set of experiments, the ratio of GFP to PEPC obtained with EV plus GFP:C11-vdXX (control) was set at a value of 1. The additional bars indicate the relative GFP/PEPC ratio for each amiRNA (as indicated) expressed with its respective GFP target. Each experiment was performed at least three times. Error bars indicate SD. The asterisks indicate statistically significant for paired t- test ( P
    Figure Legend Snippet: Validation of the predicted vd-sRNA:target mRNA complex formation by an artificial microRNA. ( A ) Duplexes predicted to be formed by complexes of amiRNAs and GFP reporter constructs containing the Chloride Channel CLC-b-like and the RPS3a-like mRNA target sequences. ( B ) N. benthamiana leaves were agro-infiltrated with (1) empty pBIN61 vector (EV) plus GFP:CLCb; (2) amiR:(+)191 plus GFP:CLCb; (3) EV plus GFP:RPS3a; and, (4) amiR:(−)69 plus GFP:RPS3a. At 3-dpi, the leaves were photographed under UV illumination. N. benthamiana leaves were agroinfiltrated in the same combinations as in ( B ). ( C ) At 3-dpi, total RNA extracts were subjected to RNA gel blot analyses with either GFP (top panel) or 7SL (lower panel) radiolabeled probes. Full size gel blots are presented in Fig. S2 . The signals in ( C ) were quantified and expressed as a ratio of the GFP to the 7SL signals. For each set of experiments, the ratio of GFP to 7SL obtained with EV plus GFP:XX (control) was set at a value of 1. The additional bars indicate the relative GFP/ 7SL ratio for each amiRNA (as indicated) expressed with its respective GFP:XX. ( D ) At 3 dpi, total protein extracts were subjected to immunoblotting with anti- GFP (top panel) and anti-PEPC (lower panel) antibodies. Full size immunoblots are presented in Fig. S3 . The immunoblot signals from ( D ) were quantified and expressed as a ratio of the GFP to the PEPC signals. For each set of experiments, the ratio of GFP to PEPC obtained with EV plus GFP:C11-vdXX (control) was set at a value of 1. The additional bars indicate the relative GFP/PEPC ratio for each amiRNA (as indicated) expressed with its respective GFP target. Each experiment was performed at least three times. Error bars indicate SD. The asterisks indicate statistically significant for paired t- test ( P

    Techniques Used: Construct, Plasmid Preparation, Western Blot

    Profiling of the vd-sRNAs recovered from PSTVd infected tomato plants and the predicted vd-sRNA:target duplexes. Sequence profiles of the (+) and the (−) PSTVd-sRNA populations recovered from the leaf tissues of infected tomato plants. Panels (A) and (B) represent the profiles of the (+) and the (−) PSTVd-I derived sRNAs, while panels (C) and (D) represent the profiles of the (+) and the (−) PSTVd-RG1 derived sRNAs. The vertical arrows denote the two vd-sRNA populations of particular interest, namely those capable of targeting potential host mRNAs: a (+) vd-sRNA located at positions 119 to 211 and a (−) vd-sRNA located at positions 69 to 49. Please note that different scales are used so as to compensate for the lower numbers of (−) vd-sRNA sequences recovered for both of the PSTVd variant infected plants. The predicted interactions between the PSTVd-sRNAs derived from the ( C ) (+) strand with the 3′ UTR of the chloride channel protein CLC-b-like mRNA and ( D ) the (−) strand with the coding region of the 40S ribosomal protein S3a-like mRNA are shown. The arrows indicate the predicted RISC mediated cleavage sites. The sequences are shown in the complementary polarity. The PairFold online tool was used to predict the minimum free energy (ΔG) secondary structures of the pairs of RNA sequences.
    Figure Legend Snippet: Profiling of the vd-sRNAs recovered from PSTVd infected tomato plants and the predicted vd-sRNA:target duplexes. Sequence profiles of the (+) and the (−) PSTVd-sRNA populations recovered from the leaf tissues of infected tomato plants. Panels (A) and (B) represent the profiles of the (+) and the (−) PSTVd-I derived sRNAs, while panels (C) and (D) represent the profiles of the (+) and the (−) PSTVd-RG1 derived sRNAs. The vertical arrows denote the two vd-sRNA populations of particular interest, namely those capable of targeting potential host mRNAs: a (+) vd-sRNA located at positions 119 to 211 and a (−) vd-sRNA located at positions 69 to 49. Please note that different scales are used so as to compensate for the lower numbers of (−) vd-sRNA sequences recovered for both of the PSTVd variant infected plants. The predicted interactions between the PSTVd-sRNAs derived from the ( C ) (+) strand with the 3′ UTR of the chloride channel protein CLC-b-like mRNA and ( D ) the (−) strand with the coding region of the 40S ribosomal protein S3a-like mRNA are shown. The arrows indicate the predicted RISC mediated cleavage sites. The sequences are shown in the complementary polarity. The PairFold online tool was used to predict the minimum free energy (ΔG) secondary structures of the pairs of RNA sequences.

    Techniques Used: Infection, Sequencing, Derivative Assay, Variant Assay

    Validation of the predicted vd-sRNA:target mRNA complex formation by an artificial microRNA. ( A ) Duplexes predicted to be formed by complexes of amiRNAs and GFP reporter constructs containing the Chloride Channel CLC-b-like and the RPS3a-like mRNA target sequences. ( B ) N. benthamiana leaves were agro-infiltrated with (1) empty pBIN61 vector (EV) plus GFP:CLCb; (2) amiR:(+)191 plus GFP:CLCb; (3) EV plus GFP:RPS3a; and, (4) amiR:(−)69 plus GFP:RPS3a. At 3-dpi, the leaves were photographed under UV illumination. N. benthamiana leaves were agroinfiltrated in the same combinations as in ( B ). ( C ) At 3-dpi, total RNA extracts were subjected to RNA gel blot analyses with either GFP (top panel) or 7SL (lower panel) radiolabeled probes. Full size gel blots are presented in Fig. S2 . The signals in ( C ) were quantified and expressed as a ratio of the GFP to the 7SL signals. For each set of experiments, the ratio of GFP to 7SL obtained with EV plus GFP:XX (control) was set at a value of 1. The additional bars indicate the relative GFP/ 7SL ratio for each amiRNA (as indicated) expressed with its respective GFP:XX. ( D ) At 3 dpi, total protein extracts were subjected to immunoblotting with anti- GFP (top panel) and anti-PEPC (lower panel) antibodies. Full size immunoblots are presented in Fig. S3 . The immunoblot signals from ( D ) were quantified and expressed as a ratio of the GFP to the PEPC signals. For each set of experiments, the ratio of GFP to PEPC obtained with EV plus GFP:C11-vdXX (control) was set at a value of 1. The additional bars indicate the relative GFP/PEPC ratio for each amiRNA (as indicated) expressed with its respective GFP target. Each experiment was performed at least three times. Error bars indicate SD. The asterisks indicate statistically significant for paired t- test ( P
    Figure Legend Snippet: Validation of the predicted vd-sRNA:target mRNA complex formation by an artificial microRNA. ( A ) Duplexes predicted to be formed by complexes of amiRNAs and GFP reporter constructs containing the Chloride Channel CLC-b-like and the RPS3a-like mRNA target sequences. ( B ) N. benthamiana leaves were agro-infiltrated with (1) empty pBIN61 vector (EV) plus GFP:CLCb; (2) amiR:(+)191 plus GFP:CLCb; (3) EV plus GFP:RPS3a; and, (4) amiR:(−)69 plus GFP:RPS3a. At 3-dpi, the leaves were photographed under UV illumination. N. benthamiana leaves were agroinfiltrated in the same combinations as in ( B ). ( C ) At 3-dpi, total RNA extracts were subjected to RNA gel blot analyses with either GFP (top panel) or 7SL (lower panel) radiolabeled probes. Full size gel blots are presented in Fig. S2 . The signals in ( C ) were quantified and expressed as a ratio of the GFP to the 7SL signals. For each set of experiments, the ratio of GFP to 7SL obtained with EV plus GFP:XX (control) was set at a value of 1. The additional bars indicate the relative GFP/ 7SL ratio for each amiRNA (as indicated) expressed with its respective GFP:XX. ( D ) At 3 dpi, total protein extracts were subjected to immunoblotting with anti- GFP (top panel) and anti-PEPC (lower panel) antibodies. Full size immunoblots are presented in Fig. S3 . The immunoblot signals from ( D ) were quantified and expressed as a ratio of the GFP to the PEPC signals. For each set of experiments, the ratio of GFP to PEPC obtained with EV plus GFP:C11-vdXX (control) was set at a value of 1. The additional bars indicate the relative GFP/PEPC ratio for each amiRNA (as indicated) expressed with its respective GFP target. Each experiment was performed at least three times. Error bars indicate SD. The asterisks indicate statistically significant for paired t- test ( P

    Techniques Used: Construct, Plasmid Preparation, Western Blot

    Effect of the PSTVd variants on the predicted target mRNAs. ( A ) Both the PSTVd-I and PSTVd-RG1 variants were inoculated into tomato plants. At 14-dpi, the plants inoculated with both of the PSTVd variants showed disease symptoms when compared to mock inoculated plants. ( B ) Total RNA extracted from tomato plants at 7, 14 and 21-dpi were used to monitor the PSTVd titer. In the graph, the dotted black line (on the X-axis) represents mock inoculated plants, while the grey and black solid lines indicate the PSTVd-I and PSTVd-RG1 inoculated plants, respectively. The effects of the PSTVd variants on the levels of the ( C ) chloride channel protein CLC-b-like and ( D ) the RPS3a-like mRNAs were evaluated at different time intervals. The expression change is presented on a log 2 scale. Each experiment was performed at least three times with true biological replicates. The changes in the expression levels of the mRNAs between the time points are shown with dotted lines. The error bars indicate SD. The asterisks indicate statistically significant for paired t- test ( P
    Figure Legend Snippet: Effect of the PSTVd variants on the predicted target mRNAs. ( A ) Both the PSTVd-I and PSTVd-RG1 variants were inoculated into tomato plants. At 14-dpi, the plants inoculated with both of the PSTVd variants showed disease symptoms when compared to mock inoculated plants. ( B ) Total RNA extracted from tomato plants at 7, 14 and 21-dpi were used to monitor the PSTVd titer. In the graph, the dotted black line (on the X-axis) represents mock inoculated plants, while the grey and black solid lines indicate the PSTVd-I and PSTVd-RG1 inoculated plants, respectively. The effects of the PSTVd variants on the levels of the ( C ) chloride channel protein CLC-b-like and ( D ) the RPS3a-like mRNAs were evaluated at different time intervals. The expression change is presented on a log 2 scale. Each experiment was performed at least three times with true biological replicates. The changes in the expression levels of the mRNAs between the time points are shown with dotted lines. The error bars indicate SD. The asterisks indicate statistically significant for paired t- test ( P

    Techniques Used: Expressing

    8) Product Images from "Nascent RNA sequencing identifies a widespread sigma70-dependent pausing regulated by Gre factors in bacteria"

    Article Title: Nascent RNA sequencing identifies a widespread sigma70-dependent pausing regulated by Gre factors in bacteria

    Journal: bioRxiv

    doi: 10.1101/2020.10.25.354225

    Statistical and in vitro biochemical analysis of G1 pauses. a, Information content (Ri) for −10LR (−10-like region) encoded by all σ 70 -Δ greAB G1 pauses as a function of its distance from TSS. The second base in the −10-like hexamer marked the location of the −10LR. The highest Ri of the hexamers ranging from −1 to +2 was adopted and assigned to −10LR (n = 3099). b, Boxplot compares the Ri of −10LR for proximal G1p and distal G1d pauses. All σ 70 promoters from RegulonDB with a labeled −10 element were used as a control (n = 950). c, d, Read length distribution at G1p and G1d pauses, respectively. Ratio of reads, number of reads with specific length(es)/number of total reads. The cartoon on the top depict the backtracked translocation states of G1d complexes based on a significant difference of their read lengths. Note, that the short ≤15-nt RNAs detected at most G1p pauses were due to the close proximity of G1p pauses to TSS that precluded determination of translocation state of G1p complexes by treatment with RNase I. e, f, RNET-seq and RNA-seq profiles of two representative genomic regions containing G1p and G1d pauses identified by RNET-seq at mraZ and yieE promoters. The first 20 nt of mraZ and yieE transcripts are shown. The red capital letters and arrows indicate the TSS and the pause peaks from RNET-seq data. g, h, In vitro validation of the σ 70 -dependent G1p/G1d pauses at mraZ and yieE promoters. The left panel shows nascent RNA in the paused complexes obtained in the presence and absence of GreA or GreB. Immobilization on streptavidin beads through 5’-biotin DNA was used to confirm integrity of the RNA-labeled paused complexes (right panel). Eσ 70 with His-tagged σ 70 was used for the assay confirming presence of σ 70 in the paused complexes. RO, run-off transcripts; St, streptavidin; Ni, Ni 2+ -NTA agarose; S, supernatant; P, pellet. i, Sequence logo for σ 70 -ΔgreAB G1p and G1d promoters and for σ 70 promoters from RegulonDB. The DNA sequences were aligned relative to the TSS. Only the strongest pause was used for analysis of the TSSs following multiple pause sites. Coordinate “0” represents TSS (commonly marked as the +1 site) in the sequence logo, otherwise the standard “+1” TSS nomenclature was used. −10R, −10 promoter element; tssR, region surrounding TSS; −10LR, −10-like region; spacer, spacing region between − 10R and TSS. j, Boxplot comparing Ri of the −10 elements for G1p (top, n = 1069) and G1d (bottom, n = 407) promoters; −10R of the same numbers of randomly chosen promoters were used as a control. k, Heatmap showing correlation between distribution of spacer length and information content (Ri) of the promoter −10 element for all σ 70 promoters (top), promoters containing G1p (middle) and G1d (bottom) pauses. The two-tailed Mann-Whitney U -test was used for the statistical analysis shown above.
    Figure Legend Snippet: Statistical and in vitro biochemical analysis of G1 pauses. a, Information content (Ri) for −10LR (−10-like region) encoded by all σ 70 -Δ greAB G1 pauses as a function of its distance from TSS. The second base in the −10-like hexamer marked the location of the −10LR. The highest Ri of the hexamers ranging from −1 to +2 was adopted and assigned to −10LR (n = 3099). b, Boxplot compares the Ri of −10LR for proximal G1p and distal G1d pauses. All σ 70 promoters from RegulonDB with a labeled −10 element were used as a control (n = 950). c, d, Read length distribution at G1p and G1d pauses, respectively. Ratio of reads, number of reads with specific length(es)/number of total reads. The cartoon on the top depict the backtracked translocation states of G1d complexes based on a significant difference of their read lengths. Note, that the short ≤15-nt RNAs detected at most G1p pauses were due to the close proximity of G1p pauses to TSS that precluded determination of translocation state of G1p complexes by treatment with RNase I. e, f, RNET-seq and RNA-seq profiles of two representative genomic regions containing G1p and G1d pauses identified by RNET-seq at mraZ and yieE promoters. The first 20 nt of mraZ and yieE transcripts are shown. The red capital letters and arrows indicate the TSS and the pause peaks from RNET-seq data. g, h, In vitro validation of the σ 70 -dependent G1p/G1d pauses at mraZ and yieE promoters. The left panel shows nascent RNA in the paused complexes obtained in the presence and absence of GreA or GreB. Immobilization on streptavidin beads through 5’-biotin DNA was used to confirm integrity of the RNA-labeled paused complexes (right panel). Eσ 70 with His-tagged σ 70 was used for the assay confirming presence of σ 70 in the paused complexes. RO, run-off transcripts; St, streptavidin; Ni, Ni 2+ -NTA agarose; S, supernatant; P, pellet. i, Sequence logo for σ 70 -ΔgreAB G1p and G1d promoters and for σ 70 promoters from RegulonDB. The DNA sequences were aligned relative to the TSS. Only the strongest pause was used for analysis of the TSSs following multiple pause sites. Coordinate “0” represents TSS (commonly marked as the +1 site) in the sequence logo, otherwise the standard “+1” TSS nomenclature was used. −10R, −10 promoter element; tssR, region surrounding TSS; −10LR, −10-like region; spacer, spacing region between − 10R and TSS. j, Boxplot comparing Ri of the −10 elements for G1p (top, n = 1069) and G1d (bottom, n = 407) promoters; −10R of the same numbers of randomly chosen promoters were used as a control. k, Heatmap showing correlation between distribution of spacer length and information content (Ri) of the promoter −10 element for all σ 70 promoters (top), promoters containing G1p (middle) and G1d (bottom) pauses. The two-tailed Mann-Whitney U -test was used for the statistical analysis shown above.

    Techniques Used: In Vitro, Labeling, Translocation Assay, RNA Sequencing Assay, Sequencing, Two Tailed Test, MANN-WHITNEY

    In vitro analysis of G1p/G1d pauses and the corresponding open promoter complexes. Protection of the nascent RNA by Eσ 70 (6His-σ 70 ) holoenzyme from digestion by RNases I and T1 at G1p (a) and G1d (b) pauses. In the regular (non-paused) elongation complex, RNAP protects in vitro 14 nt (RNase T1) and 17-18 nt (RNase I) of the 3’ RNA from the nuclease digestion 59 . The cartoons on the left show the proposed alternative translocation states of the RNA in the paused complex. The stars indicate the RNA positions labeled by [α- 32 P] UMP. c, GreB-induced transcript cleavage of nascent RNA at G1p ( mraZ ) and G1d ( yieE ) pauses. The workflow for the experiment is shown on the left. Ni, Ni 2+ -NTA beads; P, pellet. The template strand sequences of mraZ and yieE promoters and backtracked RNAs at the pause sites are shown at the bottom. Red arrows indicate the pausing peaks identified by RNET-seq. d, Permanganate footprints of the non-template and template strands of the transcription bubble at the mraZ (G1p) promoter. The positions of all T residues in the bubble are indicated. A prominent non-T band between T3 and T10 is a background of permanganate footprinting. The diagrams on the right show the transcription bubble at the mraZ promoter during G1p pausing. Black filled circles, T residues sensitive to KMnO 4 in the absence and presence of NTP; gray filled circles, permanganate-sensitive T residues in the presence of NTP; white filled circle, T residues resistant to permanganate. e, Permanganate footprints of transcription bubble at the minC (G1d) promoter. Both DNA strands of the mraZ and minC promoters including the −10R (blue), tssR/-10LR (red) elements and TSS (red capital) are shown at the bottom, and the G1p and G1d pause peaks are marked by red arrows. f, Profiles of median ChIP-seq reads coverage at G1p, G1d and control promoters based on the heatmaps (Extended data Fig. 16). g, Model depicting the structural properties of σ 70 -dependent G1p and G1d pauses. The interaction of σ 70 domains with the promoter elements, the DNA scrunching and the corresponding changes in the RNA register at G1p and G1d pauses are indicated.
    Figure Legend Snippet: In vitro analysis of G1p/G1d pauses and the corresponding open promoter complexes. Protection of the nascent RNA by Eσ 70 (6His-σ 70 ) holoenzyme from digestion by RNases I and T1 at G1p (a) and G1d (b) pauses. In the regular (non-paused) elongation complex, RNAP protects in vitro 14 nt (RNase T1) and 17-18 nt (RNase I) of the 3’ RNA from the nuclease digestion 59 . The cartoons on the left show the proposed alternative translocation states of the RNA in the paused complex. The stars indicate the RNA positions labeled by [α- 32 P] UMP. c, GreB-induced transcript cleavage of nascent RNA at G1p ( mraZ ) and G1d ( yieE ) pauses. The workflow for the experiment is shown on the left. Ni, Ni 2+ -NTA beads; P, pellet. The template strand sequences of mraZ and yieE promoters and backtracked RNAs at the pause sites are shown at the bottom. Red arrows indicate the pausing peaks identified by RNET-seq. d, Permanganate footprints of the non-template and template strands of the transcription bubble at the mraZ (G1p) promoter. The positions of all T residues in the bubble are indicated. A prominent non-T band between T3 and T10 is a background of permanganate footprinting. The diagrams on the right show the transcription bubble at the mraZ promoter during G1p pausing. Black filled circles, T residues sensitive to KMnO 4 in the absence and presence of NTP; gray filled circles, permanganate-sensitive T residues in the presence of NTP; white filled circle, T residues resistant to permanganate. e, Permanganate footprints of transcription bubble at the minC (G1d) promoter. Both DNA strands of the mraZ and minC promoters including the −10R (blue), tssR/-10LR (red) elements and TSS (red capital) are shown at the bottom, and the G1p and G1d pause peaks are marked by red arrows. f, Profiles of median ChIP-seq reads coverage at G1p, G1d and control promoters based on the heatmaps (Extended data Fig. 16). g, Model depicting the structural properties of σ 70 -dependent G1p and G1d pauses. The interaction of σ 70 domains with the promoter elements, the DNA scrunching and the corresponding changes in the RNA register at G1p and G1d pauses are indicated.

    Techniques Used: In Vitro, Translocation Assay, Labeling, Footprinting, Chromatin Immunoprecipitation

    Classification of σ 70 -dependent transcription pauses. a, Example of σ 70 -dependent pause upstream of the yjcE gene identified by RNET-seq in the σ 70 -Δ greAB strain. The genomic coordinates for 3’ ends of all uniquely mapped RNA reads (bottom lane) were determined and the read count for each 3’ end position was calculated and plotted (top lane). The genomic positions where 3’ end/3’ end median (51-bp window) read counts ratio (pause score) was ≥ 20 and read counts/10 6 reads was ≥ 10 satisfied our stringent definition for a pause site. b, Venn diagrams show the total and shared numbers of pauses identified in σ 70 -WT (n = 7412), β’-WT (n = 3543), σ 70 -Δ greAB (n = 12211) and β’-Δ greAB (n = 6498) strains. c, Distribution of σ 70 -dependent pauses among CDS, UTR, Antisense, tRNA, rRNA and ncRNA transcription in σ 70 -WT and σ 70 -Δ greAB strains. The “Antisense” pauses included those in CDS, tRNA, rRNA and ncRNA genes. d, Distribution of pause sites in promoter-proximal regions. The TSS coordinates identified by dRNA-seq 58 were used to plot pause counts against the pause distance from the nearest TSS on the same DNA strand. The zero and positive coordinates correspond to the pauses overlapping the TSS or located downstream of the TSS, respectively. The upper panel shows the counts of pauses in 50-nt bins within −2000/+2000-bp window centered at TSS. The bottom panel shows the ratio obtained by dividing count of pause sites in 5-bp sliding window to the total count of pause sites in −50/+200-bp register surrounding TSS. Heatmap (e) and mean (f) of the read counts for σ 70 -Δ greAB G1 pause sites (n = 3099) in σ 70 -ΔgreAB (left) and σ 70 -WT (right) strains. The pause sites were ranked based on the pause score (described in a ). The counts of read aligned to the sense and antisense strands in each coordinate were normalized to 0 to 1 and 0 to −1 by dividing the maximum read count in each −50/+200-bp region. The regions with multiple pause sites were counted only once. (e) . The dash line and number on the top indicate distance of the peak from TSS. The line and the shadowed region represent the mean and 95% confidence interval for the read counts ratio (f) .
    Figure Legend Snippet: Classification of σ 70 -dependent transcription pauses. a, Example of σ 70 -dependent pause upstream of the yjcE gene identified by RNET-seq in the σ 70 -Δ greAB strain. The genomic coordinates for 3’ ends of all uniquely mapped RNA reads (bottom lane) were determined and the read count for each 3’ end position was calculated and plotted (top lane). The genomic positions where 3’ end/3’ end median (51-bp window) read counts ratio (pause score) was ≥ 20 and read counts/10 6 reads was ≥ 10 satisfied our stringent definition for a pause site. b, Venn diagrams show the total and shared numbers of pauses identified in σ 70 -WT (n = 7412), β’-WT (n = 3543), σ 70 -Δ greAB (n = 12211) and β’-Δ greAB (n = 6498) strains. c, Distribution of σ 70 -dependent pauses among CDS, UTR, Antisense, tRNA, rRNA and ncRNA transcription in σ 70 -WT and σ 70 -Δ greAB strains. The “Antisense” pauses included those in CDS, tRNA, rRNA and ncRNA genes. d, Distribution of pause sites in promoter-proximal regions. The TSS coordinates identified by dRNA-seq 58 were used to plot pause counts against the pause distance from the nearest TSS on the same DNA strand. The zero and positive coordinates correspond to the pauses overlapping the TSS or located downstream of the TSS, respectively. The upper panel shows the counts of pauses in 50-nt bins within −2000/+2000-bp window centered at TSS. The bottom panel shows the ratio obtained by dividing count of pause sites in 5-bp sliding window to the total count of pause sites in −50/+200-bp register surrounding TSS. Heatmap (e) and mean (f) of the read counts for σ 70 -Δ greAB G1 pause sites (n = 3099) in σ 70 -ΔgreAB (left) and σ 70 -WT (right) strains. The pause sites were ranked based on the pause score (described in a ). The counts of read aligned to the sense and antisense strands in each coordinate were normalized to 0 to 1 and 0 to −1 by dividing the maximum read count in each −50/+200-bp region. The regions with multiple pause sites were counted only once. (e) . The dash line and number on the top indicate distance of the peak from TSS. The line and the shadowed region represent the mean and 95% confidence interval for the read counts ratio (f) .

    Techniques Used:

    -10R, spacer length and tssR/-10LR determine G1p and G1d pauses in vitro . Boxplots of pause strength for G1p (a) and G1d (b) pauses in the absence and presence of GreA or GreB. G1p ( exuR , mraZ , ileX and mocA ) and G1d ( yieE , minC , gadW , mrdB and artP ) promoters were used for the analysis. The pause strength was determined by dividing the signal intensity of run-off and paused RNA products to the signal intensity of paused RNA product in the gel for each in vitro template (Pause strength = Signal intensity[paused RNA]/(Signal intensity[paused RNA] + Signal intensity[run-off])). The pause strength in the absence of Gre factors was taken as 1 (a, b) . Boxplots show the effect on pause strength of −10R and tssR mutations in G1p promoters (c, f) , and −10R and −10LR mutations in G1d promoters (d, e) . Pause strength of the WT promoters was set to 1 (c, d, e, f) . −10R (−10LR; tssR) Ri-/Ri+, mutated −10R (−10LR; tssR) with decreased or increased Ri are indicated. The grey rectangle in each cartoon represents the motif used for mutation analysis. The original and mutated (colored in blue or red) DNA sequences designed to increase (Ri+) or decrease (Ri-) Ri are shown on the right in gene order. Two-tailed Mann-Whitney U -test was used for statistical analysis of the data. Effect of the spacer length on G1p (g) and G1d (h) pauses. The in vitro transcription was initiated on the WT template or on the mutant template with the shortened DNA spacer (left); different dinucleotide RNA primers overlapping the tssR were employed to alter the position of the TSS (right). The inset shows the run-off transcripts with higher exposure to visualize the faint bands. Structural elements of the WT and mutated promoters are shown on the bottom. Each circle represents a single nucleotide. Open blue circles, −10R; Dark red circle, overlapped nucleotide between spacer and tssR/-10LR; Open black and dark red circles, spacer; Red circles, tssR; Red and orange circles, −10LR; Filled red circle, TSS. Red arrows indicate TSS. WT, wild-type promoter; SD2, spacer with 2-nt deletion; RPS, relative pause strength. The analysis included the data from two or more independent experiments.
    Figure Legend Snippet: -10R, spacer length and tssR/-10LR determine G1p and G1d pauses in vitro . Boxplots of pause strength for G1p (a) and G1d (b) pauses in the absence and presence of GreA or GreB. G1p ( exuR , mraZ , ileX and mocA ) and G1d ( yieE , minC , gadW , mrdB and artP ) promoters were used for the analysis. The pause strength was determined by dividing the signal intensity of run-off and paused RNA products to the signal intensity of paused RNA product in the gel for each in vitro template (Pause strength = Signal intensity[paused RNA]/(Signal intensity[paused RNA] + Signal intensity[run-off])). The pause strength in the absence of Gre factors was taken as 1 (a, b) . Boxplots show the effect on pause strength of −10R and tssR mutations in G1p promoters (c, f) , and −10R and −10LR mutations in G1d promoters (d, e) . Pause strength of the WT promoters was set to 1 (c, d, e, f) . −10R (−10LR; tssR) Ri-/Ri+, mutated −10R (−10LR; tssR) with decreased or increased Ri are indicated. The grey rectangle in each cartoon represents the motif used for mutation analysis. The original and mutated (colored in blue or red) DNA sequences designed to increase (Ri+) or decrease (Ri-) Ri are shown on the right in gene order. Two-tailed Mann-Whitney U -test was used for statistical analysis of the data. Effect of the spacer length on G1p (g) and G1d (h) pauses. The in vitro transcription was initiated on the WT template or on the mutant template with the shortened DNA spacer (left); different dinucleotide RNA primers overlapping the tssR were employed to alter the position of the TSS (right). The inset shows the run-off transcripts with higher exposure to visualize the faint bands. Structural elements of the WT and mutated promoters are shown on the bottom. Each circle represents a single nucleotide. Open blue circles, −10R; Dark red circle, overlapped nucleotide between spacer and tssR/-10LR; Open black and dark red circles, spacer; Red circles, tssR; Red and orange circles, −10LR; Filled red circle, TSS. Red arrows indicate TSS. WT, wild-type promoter; SD2, spacer with 2-nt deletion; RPS, relative pause strength. The analysis included the data from two or more independent experiments.

    Techniques Used: In Vitro, Mutagenesis, Two Tailed Test, MANN-WHITNEY

    9) Product Images from "The genomic organization and expression pattern of the low-affinity Fc gamma receptors (FcγR) in the Göttingen minipig"

    Article Title: The genomic organization and expression pattern of the low-affinity Fc gamma receptors (FcγR) in the Göttingen minipig

    Journal: Immunogenetics

    doi: 10.1007/s00251-018-01099-1

    Single-cell RNA sequencing analysis of FCGR expression in minipig, human, and mouse PBMCs. For every species, the cells were clustered individually according to their gene expression pattern and displayed as dot plots by dimensionality reduction using t-SNE. The clustering for every species is shown on the left with outlines for better separation. Individual clusters are labeled with “Mo” for monocytes, “DC” for dendritic cells, “NK” for NK cells, “CTL” for cytotoxic T lymphocytes, “T” for T cells, “B” for B cells, and “?” for mixture cell types. In mouse PBMCs, monocytes and dendritic cells are summarized in the “Mo/DC” cluster. The visualization shows the expression of the FCGR indicated above where positive cells are labeled in blue and negative cells in gray
    Figure Legend Snippet: Single-cell RNA sequencing analysis of FCGR expression in minipig, human, and mouse PBMCs. For every species, the cells were clustered individually according to their gene expression pattern and displayed as dot plots by dimensionality reduction using t-SNE. The clustering for every species is shown on the left with outlines for better separation. Individual clusters are labeled with “Mo” for monocytes, “DC” for dendritic cells, “NK” for NK cells, “CTL” for cytotoxic T lymphocytes, “T” for T cells, “B” for B cells, and “?” for mixture cell types. In mouse PBMCs, monocytes and dendritic cells are summarized in the “Mo/DC” cluster. The visualization shows the expression of the FCGR indicated above where positive cells are labeled in blue and negative cells in gray

    Techniques Used: RNA Sequencing Assay, Expressing, Labeling

    10) Product Images from "Contrasting Sex-and Caste-Dependent piRNA Profiles in the Transposon Depleted Haplodiploid Honeybee Apis mellifera"

    Article Title: Contrasting Sex-and Caste-Dependent piRNA Profiles in the Transposon Depleted Haplodiploid Honeybee Apis mellifera

    Journal: Genome Biology and Evolution

    doi: 10.1093/gbe/evx087

    —Candidate piRNAs in the honeybee genome. Shaded regions show the minimum/maximum values observed between biological replicates. ( A ) Size distribution of small RNAs of 24 bp and larger mapped to the honeybee genome. A peak between 26 and 31 nt can be observed, consistent with the size of piRNAs in other species. ( B ) Percentage of sequences with a 5′ U. The majority of sequences between 26 and 31 nt have the 5′ U characteristic of piRNAs. ( C ) Size distribution of small RNAs mapped to transposons, the typical piRNA peak between 26 and 31 nt is more pronounced than for the total small RNA population.
    Figure Legend Snippet: —Candidate piRNAs in the honeybee genome. Shaded regions show the minimum/maximum values observed between biological replicates. ( A ) Size distribution of small RNAs of 24 bp and larger mapped to the honeybee genome. A peak between 26 and 31 nt can be observed, consistent with the size of piRNAs in other species. ( B ) Percentage of sequences with a 5′ U. The majority of sequences between 26 and 31 nt have the 5′ U characteristic of piRNAs. ( C ) Size distribution of small RNAs mapped to transposons, the typical piRNA peak between 26 and 31 nt is more pronounced than for the total small RNA population.

    Techniques Used:

    11) Product Images from "Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs"

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs

    Journal: Nature Communications

    doi: 10.1038/s41467-018-02866-0

    Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)
    Figure Legend Snippet: Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Techniques Used: Synthesized, Activity Assay, Amplification, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Whisker Assay

    12) Product Images from "Short non-coding RNA fragments accumulating in chloroplasts: footprints of RNA binding proteins?"

    Article Title: Short non-coding RNA fragments accumulating in chloroplasts: footprints of RNA binding proteins?

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1138

    Mapping of transcript ends in the vicinity of selected sRNAs (A = rps15 5′; B = rps12 5′; C = psbD-psbC; D = rps7-ndhB). Total Arabidopsis RNA was ligated with RNA oligos selectively either at the 3′- or at the 5′-end, reverse-transcribed and amplified by PCR with combinations of gene-specific and oligo-specific primers.The amplification products were separated on an agarose gel (left side of each panel). PCR products were gel-purified and cloned. Clones were selected and sequenced. The last base before the sequence of the RNA oligo corresponds to the end of the original chloroplast RNA ligated. These ends are indicated by open arrowheads (for 5′-RACE experiments) or by filled arrowheads (for 3′-RACE experiments) above a blowup of a sequence stretch covering parts of the intergenic region containing the sRNAs (upper case) at the center. The numbers above the arrowheads point out numbers of clones that correspond to a particular transcript end. In case of 5′-RACE, the numbers refer to independent clones as evidenced by different bar-codes introduced via randomized nucleotides in the ligated 5′-RNA oligo. Number of clones indicating independent ends outside of the blowup region are indicated above outward-facing arrows at the ends of the sequence shown here. All further symbols and numbers are explained in Figure 1 .
    Figure Legend Snippet: Mapping of transcript ends in the vicinity of selected sRNAs (A = rps15 5′; B = rps12 5′; C = psbD-psbC; D = rps7-ndhB). Total Arabidopsis RNA was ligated with RNA oligos selectively either at the 3′- or at the 5′-end, reverse-transcribed and amplified by PCR with combinations of gene-specific and oligo-specific primers.The amplification products were separated on an agarose gel (left side of each panel). PCR products were gel-purified and cloned. Clones were selected and sequenced. The last base before the sequence of the RNA oligo corresponds to the end of the original chloroplast RNA ligated. These ends are indicated by open arrowheads (for 5′-RACE experiments) or by filled arrowheads (for 3′-RACE experiments) above a blowup of a sequence stretch covering parts of the intergenic region containing the sRNAs (upper case) at the center. The numbers above the arrowheads point out numbers of clones that correspond to a particular transcript end. In case of 5′-RACE, the numbers refer to independent clones as evidenced by different bar-codes introduced via randomized nucleotides in the ligated 5′-RNA oligo. Number of clones indicating independent ends outside of the blowup region are indicated above outward-facing arrows at the ends of the sequence shown here. All further symbols and numbers are explained in Figure 1 .

    Techniques Used: Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Purification, Clone Assay, Sequencing

    13) Product Images from "A comparative analysis of library prep approaches for sequencing low input translatome samples"

    Article Title: A comparative analysis of library prep approaches for sequencing low input translatome samples

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-5066-2

    Hierarchical clustering of expression levels, based on the rank of the count of exon per million mapped reads (CPM). Dendrogram represents Spearman correlation coefficients between pairs of samples. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples.. Color scale: Spearman correlation coefficients
    Figure Legend Snippet: Hierarchical clustering of expression levels, based on the rank of the count of exon per million mapped reads (CPM). Dendrogram represents Spearman correlation coefficients between pairs of samples. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples.. Color scale: Spearman correlation coefficients

    Techniques Used: Expressing

    Hierarchical clustering based on the rank of IP/input value. Dendrogram represents Spearman correlation coefficients between pairs of samples. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples
    Figure Legend Snippet: Hierarchical clustering based on the rank of IP/input value. Dendrogram represents Spearman correlation coefficients between pairs of samples. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples

    Techniques Used:

    Descriptive characteristic of enrichment or depletion profiles as generated by the different library preparation kits. Genes which have at least 20 raw reads in the input samples and a ratio of IP/Input ≥2 or Input/IP ≥2 were used to generate the plots. a Total number of transcripts enriched or depleted. b Percentage of enriched or depleted transcripts grouped into different bins. X-axis: log2(IP/input), Y-axis: percentage of genes in each bin over whole population. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples
    Figure Legend Snippet: Descriptive characteristic of enrichment or depletion profiles as generated by the different library preparation kits. Genes which have at least 20 raw reads in the input samples and a ratio of IP/Input ≥2 or Input/IP ≥2 were used to generate the plots. a Total number of transcripts enriched or depleted. b Percentage of enriched or depleted transcripts grouped into different bins. X-axis: log2(IP/input), Y-axis: percentage of genes in each bin over whole population. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples

    Techniques Used: Generated

    Enrichment profiles and top 50 enriched transcripts. a Enrichment factor of transcripts are sorted in decreasing order based on log2 (IP/input). X-axis:transcripts, Y-axis:log2 value of enrichment (IP/Input). b Boxplot of top 50 enriched transcripts. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples
    Figure Legend Snippet: Enrichment profiles and top 50 enriched transcripts. a Enrichment factor of transcripts are sorted in decreasing order based on log2 (IP/input). X-axis:transcripts, Y-axis:log2 value of enrichment (IP/Input). b Boxplot of top 50 enriched transcripts. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples

    Techniques Used:

    Descriptive characteristics of raw and mapped reads. a Total number of raw reads and number of reads mapped to the mouse genome (mm10, GRCm38.84). b Percentage of reads mapped to exonic, intronic and intergenic regions. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA
    Figure Legend Snippet: Descriptive characteristics of raw and mapped reads. a Total number of raw reads and number of reads mapped to the mouse genome (mm10, GRCm38.84). b Percentage of reads mapped to exonic, intronic and intergenic regions. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA

    Techniques Used:

    Distribution of normalized mean expression of the first (last) 100 bases of transcripts (in 5′- > 3′-orientation). X axis represents the 5′-3′ normalized position; Y axis represents normalized coverage. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuG samples; Green and grey: TruSeq samples. Solid: Input samples. Dotted: Ribo-IP samples
    Figure Legend Snippet: Distribution of normalized mean expression of the first (last) 100 bases of transcripts (in 5′- > 3′-orientation). X axis represents the 5′-3′ normalized position; Y axis represents normalized coverage. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuG samples; Green and grey: TruSeq samples. Solid: Input samples. Dotted: Ribo-IP samples

    Techniques Used: Expressing

    Venn diagrams of identified features in the different libraries. The features with CPM ≥ 1 in at least one out of 3 replicates were used to generate these plots. a and c represent input samples and b and d represent IP samples. Most transcripts were detected by all kits tested. However, a higher rate of agreement is seen between the NEB, TruSeq and SMART-Seq prepared samples. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA
    Figure Legend Snippet: Venn diagrams of identified features in the different libraries. The features with CPM ≥ 1 in at least one out of 3 replicates were used to generate these plots. a and c represent input samples and b and d represent IP samples. Most transcripts were detected by all kits tested. However, a higher rate of agreement is seen between the NEB, TruSeq and SMART-Seq prepared samples. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA

    Techniques Used:

    14) Product Images from "High-throughput sequencing reveals circular substrates for an archaeal RNA ligase"

    Article Title: High-throughput sequencing reveals circular substrates for an archaeal RNA ligase

    Journal: RNA Biology

    doi: 10.1080/15476286.2017.1302640

    Pab 1020 RNA ligase binds single-stranded DNA and RNA, but only circularizes single-stranded RNA oligonucleotides. (A) EMSA assays were performed with internally labeled (Cy5) single stranded DNA or RNA oligonucleotides using increasing amounts of wild-type (WT) Pab 1020 RNA ligase. The relative amount of bound DNA or RNA was plotted against the protein concentration. Insert: On the EMSA gel, the amount of the higher molecular weight bands, corresponding to Pab 1020-nucleic acid complexes, increased as a function of the protein concentration. (B) RNA and DNA ligation assays with WT and mutant K95G of Pab 1020 RNA ligase. Standard ligation reactions containing 10 pmol Cy5-RNA or -DNA molecules and 200 pmol RNA ligase Pab 1020 were incubated 90 min at 50°C. Reaction products were resolved on denaturing PAGE and a 700 nm scan of the gel was performed on Licor Odyssey Infrared Imager. While no activity was observed with DNA substrate, Pab 1020 RNA ligase circularized an RNA oligoribonucleotide as shown on the gel with the apparition of a lower band corresponding to circular RNA molecules. Expectedly, a control reaction with an inactive enzyme (mutant K95G) presented no lower band. (C) Identical to panel (A), except that the enzymes used in the EMSA assays corresponded to the mutant G296A (dimerization domain) and the amino-terminal domain of 250 residues carrying a nucleotide transferase (NTase) domain. Both mutants were able to form RNA-Protein complexes with 18-mers single-stranded RNA. (D) Identical to panel (B), except that circularization was performed only with RNA substrate and with G296A mutant and NTase domain. No circRNAs were observed (positive control is indicated in panel B).
    Figure Legend Snippet: Pab 1020 RNA ligase binds single-stranded DNA and RNA, but only circularizes single-stranded RNA oligonucleotides. (A) EMSA assays were performed with internally labeled (Cy5) single stranded DNA or RNA oligonucleotides using increasing amounts of wild-type (WT) Pab 1020 RNA ligase. The relative amount of bound DNA or RNA was plotted against the protein concentration. Insert: On the EMSA gel, the amount of the higher molecular weight bands, corresponding to Pab 1020-nucleic acid complexes, increased as a function of the protein concentration. (B) RNA and DNA ligation assays with WT and mutant K95G of Pab 1020 RNA ligase. Standard ligation reactions containing 10 pmol Cy5-RNA or -DNA molecules and 200 pmol RNA ligase Pab 1020 were incubated 90 min at 50°C. Reaction products were resolved on denaturing PAGE and a 700 nm scan of the gel was performed on Licor Odyssey Infrared Imager. While no activity was observed with DNA substrate, Pab 1020 RNA ligase circularized an RNA oligoribonucleotide as shown on the gel with the apparition of a lower band corresponding to circular RNA molecules. Expectedly, a control reaction with an inactive enzyme (mutant K95G) presented no lower band. (C) Identical to panel (A), except that the enzymes used in the EMSA assays corresponded to the mutant G296A (dimerization domain) and the amino-terminal domain of 250 residues carrying a nucleotide transferase (NTase) domain. Both mutants were able to form RNA-Protein complexes with 18-mers single-stranded RNA. (D) Identical to panel (B), except that circularization was performed only with RNA substrate and with G296A mutant and NTase domain. No circRNAs were observed (positive control is indicated in panel B).

    Techniques Used: Labeling, Protein Concentration, Molecular Weight, DNA Ligation, Mutagenesis, Ligation, Incubation, Polyacrylamide Gel Electrophoresis, Activity Assay, Positive Control

    Pab 1020 RNA ligase circularizes physiologically relevant RNA molecules. (A) RNA binding between Pab 1020 RNA ligase (0.2 to 4.5 μM) and the in vitro transcripts (0.4 μM) corresponding to BoxC/D RNAs SR4 (▪) and SR29 (▴) and 5S rRNA (▾) was analyzed by EMSA. A fraction of protein-RNA complex formed was plotted as a function of input protein. Insert: On the EMSA gel, the amount of the higher molecular weight bands, corresponding to Pab 1020-nucleic acid complexes, increased as a function of the protein concentration. (B) In vitro transcript of 5S rRNA was incubated (right panel) or not (left panel) with Pab 1020 RNA ligase (WT) for 120 min at 55°C. After incubation, recovered RNAs were treated or not with exoribonuclease RNase R for 120 min at 37°C before analysis on a 7% acrylamide 8M urea gel. (C) Schematic illustration of RT-PCR experiments on linear and circular RNAs with divergent primers to distinguish linear RNAs from circular RNAs products after incubation with Pab 1020 RNA ligase. Only reverse transcription and PCR reactions on a circular RNA template will lead to the total amplification of the substrate sequence. (D) cDNA generated using outward facing primers on RNAs previously incubated (+) or not (−) with Pab 1020 RNA ligase and in the presence (+) or absence (−) of RNase R were separated by gel electrophoresis. A full-length product attesting to amplification of circular RNA molecules, indicated by the asterisk, was observed for 5S rRNA (128 bp), Box C/D SR4 RNA (68 bp) and Box C/D SR29 RNA (66 bp). Circularization was observed only in the presence of Pab 1020 RNA ligase.
    Figure Legend Snippet: Pab 1020 RNA ligase circularizes physiologically relevant RNA molecules. (A) RNA binding between Pab 1020 RNA ligase (0.2 to 4.5 μM) and the in vitro transcripts (0.4 μM) corresponding to BoxC/D RNAs SR4 (▪) and SR29 (▴) and 5S rRNA (▾) was analyzed by EMSA. A fraction of protein-RNA complex formed was plotted as a function of input protein. Insert: On the EMSA gel, the amount of the higher molecular weight bands, corresponding to Pab 1020-nucleic acid complexes, increased as a function of the protein concentration. (B) In vitro transcript of 5S rRNA was incubated (right panel) or not (left panel) with Pab 1020 RNA ligase (WT) for 120 min at 55°C. After incubation, recovered RNAs were treated or not with exoribonuclease RNase R for 120 min at 37°C before analysis on a 7% acrylamide 8M urea gel. (C) Schematic illustration of RT-PCR experiments on linear and circular RNAs with divergent primers to distinguish linear RNAs from circular RNAs products after incubation with Pab 1020 RNA ligase. Only reverse transcription and PCR reactions on a circular RNA template will lead to the total amplification of the substrate sequence. (D) cDNA generated using outward facing primers on RNAs previously incubated (+) or not (−) with Pab 1020 RNA ligase and in the presence (+) or absence (−) of RNase R were separated by gel electrophoresis. A full-length product attesting to amplification of circular RNA molecules, indicated by the asterisk, was observed for 5S rRNA (128 bp), Box C/D SR4 RNA (68 bp) and Box C/D SR29 RNA (66 bp). Circularization was observed only in the presence of Pab 1020 RNA ligase.

    Techniques Used: RNA Binding Assay, In Vitro, Molecular Weight, Protein Concentration, Incubation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Sequencing, Generated, Nucleic Acid Electrophoresis

    15) Product Images from "Regulation of the Neurospora Circadian Clock by the Spliceosome Component PRP5"

    Article Title: Regulation of the Neurospora Circadian Clock by the Spliceosome Component PRP5

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.119.400500

    PRP5 regulates Neurospora circadian rhythms. (A) qRT-PCR results of frq , wc-1 and wc-2 in ds control and ds prp5 strains. The strains were grown in constant light (LL). The expression was normalized to 28s rRNA. Values are mean ± SD, n = 5. (B) Western blot results of FRQ, WC-1 and WC-2 in ds control and ds prp5 strains. The strains were grown in LL. Values are mean ± SD, n = 5. (C) qRT-PCR analysis showing the expression of frq RNA in ds prp5 in constant dark over a 48-h time course. Electrophoresis results of RNA samples were shown as control. The expression was normalized to 28s rRNA. The values are presented as the mean ± SD, n = 3. (D) Western blot analysis of the FRQ protein levels in ds prp5 in constant darkness over a 48-h time course. The values are presented as the mean ± SD, n = 3. (E) Representative results of luciferase reporter assays showing the frq promoter activity of the indicated strains in constant darkness. The measurement of luciferase activity was normalized by subtracting the baseline luciferase signal.
    Figure Legend Snippet: PRP5 regulates Neurospora circadian rhythms. (A) qRT-PCR results of frq , wc-1 and wc-2 in ds control and ds prp5 strains. The strains were grown in constant light (LL). The expression was normalized to 28s rRNA. Values are mean ± SD, n = 5. (B) Western blot results of FRQ, WC-1 and WC-2 in ds control and ds prp5 strains. The strains were grown in LL. Values are mean ± SD, n = 5. (C) qRT-PCR analysis showing the expression of frq RNA in ds prp5 in constant dark over a 48-h time course. Electrophoresis results of RNA samples were shown as control. The expression was normalized to 28s rRNA. The values are presented as the mean ± SD, n = 3. (D) Western blot analysis of the FRQ protein levels in ds prp5 in constant darkness over a 48-h time course. The values are presented as the mean ± SD, n = 3. (E) Representative results of luciferase reporter assays showing the frq promoter activity of the indicated strains in constant darkness. The measurement of luciferase activity was normalized by subtracting the baseline luciferase signal.

    Techniques Used: Quantitative RT-PCR, Expressing, Western Blot, Electrophoresis, Luciferase, Activity Assay

    16) Product Images from "Enhancer Associated Long Non-coding RNA Transcription and Gene Regulation in Experimental Models of Rickettsial Infection"

    Article Title: Enhancer Associated Long Non-coding RNA Transcription and Gene Regulation in Experimental Models of Rickettsial Infection

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.03014

    Analysis of epigenomic signatures around transcription start site (TSS) of elncRNAs and plncRNAs. (A–D) Average normalized RPKM (reads per kilobase million) values of RNA polII, p300, DNaseI hypersensitivity site, and CCCTC binding factor (CTCF) in elncRNAs and plncRNAs, respectively; (E,F) Contrast chromatin (H3K4Me1 and H3K4Me3) and epigenetic (RNA polII, p300, DNaseI hypersensitivity and CTCF binding site) landscapes (in mouse lungs) around TSS of NONMMUT013718 and NONMMUT024103 elncRNAs, respectively. *** P ≤ 0.001 and ns = non-significant.
    Figure Legend Snippet: Analysis of epigenomic signatures around transcription start site (TSS) of elncRNAs and plncRNAs. (A–D) Average normalized RPKM (reads per kilobase million) values of RNA polII, p300, DNaseI hypersensitivity site, and CCCTC binding factor (CTCF) in elncRNAs and plncRNAs, respectively; (E,F) Contrast chromatin (H3K4Me1 and H3K4Me3) and epigenetic (RNA polII, p300, DNaseI hypersensitivity and CTCF binding site) landscapes (in mouse lungs) around TSS of NONMMUT013718 and NONMMUT024103 elncRNAs, respectively. *** P ≤ 0.001 and ns = non-significant.

    Techniques Used: Binding Assay

    17) Product Images from "MDR1 mediated chemoresistance: BMI1 and TIP60 in action"

    Article Title: MDR1 mediated chemoresistance: BMI1 and TIP60 in action

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbagrm.2016.06.002

    MDR1 promoter is regulated by Cisplatin and BMI1 (A) Schema of the proximal and distal promoter region of MDR1 is shown in the top panel. The arrows represent a single reverse primer and three different forward primer binding sites used to amplify the E1–E2, E1a–E2 or the E1b–E2 amplicon. Lower panel depicts the E1b–E2 or the 18S rRNA (internal control) PCR amplicon. OVCAR4 and CP20 cells were first transfected with scrambled-control (CsiRNA) or BMI1siRNA treated with 4µM (OVCAR4) or 10µM (CP20) cisplatin for 48h or left untreated. RNA isolated from treated and transfected cells were reverse transcribed into cDNA and PCR performed using primers as indicated. Numbers below the lane represent relative densitometry of the E1b–E2 amplicon determined using NIH ImageJ. (B) A representative schema of the E-box clusters (S1–S8) within the proximal MDR1 promoter is shown in the upper panel. The major transcription start site (+1) is indicated by an arrow. Lower left panel represents the sites of BMI1 association within the regulatory regions of the MDR1 promoter as determined by the ChIP Assay. OVCAR4 and CP20 cells were treated with cisplatin (4µM OVCAR4, 10µM CP20) and 24µM NU9056 (TIP60 inhibitor) either alone or in combination for 48h. Immunoprecipitation of the sheared DNA was performed with BMI1 antibody and DNA was extracted to perform PCR with primers that specifically amplify either S1–S2, S3–S6 or S7–S8 regions as indicated in the schema. In the lower right panel, densitometry was performed using S3/6 amplicon signals from ChIP BMI1 (normalized to respective input) and graphically presented with mean values ± SD. Values were obtained from three independent experiments and data was analyzed using ANOVA with Dunnett’s method for multiple comparisons,* denotes p -value
    Figure Legend Snippet: MDR1 promoter is regulated by Cisplatin and BMI1 (A) Schema of the proximal and distal promoter region of MDR1 is shown in the top panel. The arrows represent a single reverse primer and three different forward primer binding sites used to amplify the E1–E2, E1a–E2 or the E1b–E2 amplicon. Lower panel depicts the E1b–E2 or the 18S rRNA (internal control) PCR amplicon. OVCAR4 and CP20 cells were first transfected with scrambled-control (CsiRNA) or BMI1siRNA treated with 4µM (OVCAR4) or 10µM (CP20) cisplatin for 48h or left untreated. RNA isolated from treated and transfected cells were reverse transcribed into cDNA and PCR performed using primers as indicated. Numbers below the lane represent relative densitometry of the E1b–E2 amplicon determined using NIH ImageJ. (B) A representative schema of the E-box clusters (S1–S8) within the proximal MDR1 promoter is shown in the upper panel. The major transcription start site (+1) is indicated by an arrow. Lower left panel represents the sites of BMI1 association within the regulatory regions of the MDR1 promoter as determined by the ChIP Assay. OVCAR4 and CP20 cells were treated with cisplatin (4µM OVCAR4, 10µM CP20) and 24µM NU9056 (TIP60 inhibitor) either alone or in combination for 48h. Immunoprecipitation of the sheared DNA was performed with BMI1 antibody and DNA was extracted to perform PCR with primers that specifically amplify either S1–S2, S3–S6 or S7–S8 regions as indicated in the schema. In the lower right panel, densitometry was performed using S3/6 amplicon signals from ChIP BMI1 (normalized to respective input) and graphically presented with mean values ± SD. Values were obtained from three independent experiments and data was analyzed using ANOVA with Dunnett’s method for multiple comparisons,* denotes p -value

    Techniques Used: Binding Assay, Amplification, Polymerase Chain Reaction, Transfection, Isolation, Chromatin Immunoprecipitation, Immunoprecipitation

    18) Product Images from "Neurotoxicity of HIV-1 Tat is attributed to its penetrating property"

    Article Title: Neurotoxicity of HIV-1 Tat is attributed to its penetrating property

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-70950-x

    Tat or Exo-Tat mRNA levels in mouse organs. The cDNA sequence encoding Exo-Tat was subcloned into an adeno-associated virus vector pAAV-MCS. Subsequenced vector pAAV-Tat or pAAV-Exo-Tat was co-transfected with pAAV-DJ and pHelper at 1:1:1 ratio into HEK293T cells to generate AAV-Tat or AAV-Exo-Tat viruses. AAV viruses (1 × 10 12 GC in 200 μl PBS) were injected into mice via tail vein. Three weeks later, the mice were anesthetized with isoflurane and blood was drawn by cardiac puncture. Brain, heart, intestine, kidney, liver, lung, muscle and spleen were taken immediately. Total RNA was extracted from each organ using Trizol reagent. mRNA levels were measured by qRT-PCR. The mean relative copy number of 3 mice is shown here.
    Figure Legend Snippet: Tat or Exo-Tat mRNA levels in mouse organs. The cDNA sequence encoding Exo-Tat was subcloned into an adeno-associated virus vector pAAV-MCS. Subsequenced vector pAAV-Tat or pAAV-Exo-Tat was co-transfected with pAAV-DJ and pHelper at 1:1:1 ratio into HEK293T cells to generate AAV-Tat or AAV-Exo-Tat viruses. AAV viruses (1 × 10 12 GC in 200 μl PBS) were injected into mice via tail vein. Three weeks later, the mice were anesthetized with isoflurane and blood was drawn by cardiac puncture. Brain, heart, intestine, kidney, liver, lung, muscle and spleen were taken immediately. Total RNA was extracted from each organ using Trizol reagent. mRNA levels were measured by qRT-PCR. The mean relative copy number of 3 mice is shown here.

    Techniques Used: Sequencing, Plasmid Preparation, Transfection, Injection, Mouse Assay, Quantitative RT-PCR

    19) Product Images from "Homeostatic control of Argonaute stability by microRNA availability"

    Article Title: Homeostatic control of Argonaute stability by microRNA availability

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.2606

    Accumulation of mouse and Drosophila Argonautes is dependent on loading with functional small RNAs (a) Dicer −/− cells were transfected with tRNA-mir-451 or tRNA-mir-155 fusion constructs, and RNAs recovered from mAgo2-IP were subjected to Northern blotting. (b) RNA samples from (a) were subjected to 5'-end labelling to visualize all RNAs. Signals in mAgo2-IP derived mostly from mature miR-451 and pre-mir-155 . (c) Western blot showing that mAgo2 was stabilized in cells expressing wild-type Dicer-independent mir-451 , but not in cells expressing Dicer-dependent mir-155 or tRNA[lys] parent construct. (d) A tRNA-mir-451 cleavage mutant (CM) contains bulged nucleotides at its mAgo2 cleavage site. This arrests its processing as a pre-miRNA hairpin, as confirmed by Northern blotting of input and mAgo2-IP material from transfected Dicer −/− cells. Note that cells expressing wild-type tRNA-mir-451 mildly accumulate the hairpin precursor, but mAgo2 complexes strictly contain cleaved and matured forms of miR-451. (e) mAgo2 was not stabilized by tRNA-mir-451-CM , compared to tRNA-mir-451 . (f) Luciferase sensor assays in Drosophila S2 cells. Sensor constructs were empty, or contained complementary sites to miR-276a-5p or miR-276a-3p. Tests were done in quadruplet for each sample. Error bars represent standard variation. Student’s two-tailed, equal variance t-test was performed. (g) S2 cells were depleted of miRNA biogenesis factors using the indicated dsRNAs or control GFP dsRNA. Transfection of scrambled siRNA duplex mildly improved AGO1 accumulation, but synthetic miR-276a-miR-276a* duplex induced robust recovery of AGO1 levels. See also Fig. S6 .
    Figure Legend Snippet: Accumulation of mouse and Drosophila Argonautes is dependent on loading with functional small RNAs (a) Dicer −/− cells were transfected with tRNA-mir-451 or tRNA-mir-155 fusion constructs, and RNAs recovered from mAgo2-IP were subjected to Northern blotting. (b) RNA samples from (a) were subjected to 5'-end labelling to visualize all RNAs. Signals in mAgo2-IP derived mostly from mature miR-451 and pre-mir-155 . (c) Western blot showing that mAgo2 was stabilized in cells expressing wild-type Dicer-independent mir-451 , but not in cells expressing Dicer-dependent mir-155 or tRNA[lys] parent construct. (d) A tRNA-mir-451 cleavage mutant (CM) contains bulged nucleotides at its mAgo2 cleavage site. This arrests its processing as a pre-miRNA hairpin, as confirmed by Northern blotting of input and mAgo2-IP material from transfected Dicer −/− cells. Note that cells expressing wild-type tRNA-mir-451 mildly accumulate the hairpin precursor, but mAgo2 complexes strictly contain cleaved and matured forms of miR-451. (e) mAgo2 was not stabilized by tRNA-mir-451-CM , compared to tRNA-mir-451 . (f) Luciferase sensor assays in Drosophila S2 cells. Sensor constructs were empty, or contained complementary sites to miR-276a-5p or miR-276a-3p. Tests were done in quadruplet for each sample. Error bars represent standard variation. Student’s two-tailed, equal variance t-test was performed. (g) S2 cells were depleted of miRNA biogenesis factors using the indicated dsRNAs or control GFP dsRNA. Transfection of scrambled siRNA duplex mildly improved AGO1 accumulation, but synthetic miR-276a-miR-276a* duplex induced robust recovery of AGO1 levels. See also Fig. S6 .

    Techniques Used: Functional Assay, Transfection, Construct, Northern Blot, Derivative Assay, Western Blot, Expressing, Mutagenesis, Luciferase, Two Tailed Test

    20) Product Images from "CGGBP1 regulates cell cycle in cancer cells"

    Article Title: CGGBP1 regulates cell cycle in cancer cells

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-12-28

    CGGBP1 depletion in U-2987 MG cells is associated with increased expression of CDKN1A and GAS1 . A: A strong decrease in CGGBP1 mRNA expression shows the efficiency of siRNA transfections. Using GAPDH as control, increase in the mRNA levels of CDKN1A and GAS1 was seen. RNA samples from duplicate transfections were pooled and analyzed in triplicates and relative change in expression calculated by delta delta Ct method. All changes in expression are significant between control siRNA and CGGBP1 siRNA treatments (p
    Figure Legend Snippet: CGGBP1 depletion in U-2987 MG cells is associated with increased expression of CDKN1A and GAS1 . A: A strong decrease in CGGBP1 mRNA expression shows the efficiency of siRNA transfections. Using GAPDH as control, increase in the mRNA levels of CDKN1A and GAS1 was seen. RNA samples from duplicate transfections were pooled and analyzed in triplicates and relative change in expression calculated by delta delta Ct method. All changes in expression are significant between control siRNA and CGGBP1 siRNA treatments (p

    Techniques Used: Expressing, Transfection

    21) Product Images from "The deubiquitinase Usp9x regulates PRC2-mediated chromatin reprogramming during mouse development"

    Article Title: The deubiquitinase Usp9x regulates PRC2-mediated chromatin reprogramming during mouse development

    Journal: Nature Communications

    doi: 10.1038/s41467-021-21910-0

    Usp9x -mutant embryos arrest at E9.5–E11.5 and display defective repression of early lineage programs marked by H3K27me3. a Genetic cross to delete Usp9x in epiblast derivatives of postimplantation embryos. Quantification of recovered (live) male embryos at several postimplantation stages (right). b Sample images and quantification of control and mutant embryo phenotypes. Relative to controls (left), E9.5 embryos show variable developmental delay, with closed arrow indicating an open anterior neuropore. E11.5 embryos show a range of phenotypes, from hemorrhage to severe delay and death (tally includes dead embryos). Open arrow indicates pericardial edema. Scale bars = 250 µm (E9.5), 2.8 mm (E11.5), with N indicated. c MA plots of expression changes by RNA-seq in two litters of Usp9x mutants versus controls (at E8.5). 3 mutants and 3 controls were sequenced per litter ( N = 12 embryos total; see Supplementary Fig. 3b-d ). d Enrichr analysis of the top-enriched transcription factors (TF) that bind to the genes upregulated in Usp9x -mutant embryos in various cell types. e Expression of the 71 genes upregulated in Usp9x mutants during wild-type development 56 . FC , Fold-change relative to E6.5 embryos. f Distribution and boxplot quantification of H3K27me3 levels 59 over the promoters of genes upregulated in Usp9x mutants (10 kb upstream, 1 kb downstream of TSS). g Representative genome browser tracks of H3K27me3 in wild-type embryos (E6.5–E8.5) at the Nodal locus 59 . Known enhancer elements are highlighted and show gains of H3K27me3. h Nodal mRNA expression in wild-type development 56 . i Nodal mRNA expression in E8.5 Usp9x- mutant or control embryos. Boxplot hinges show the first and third quartiles, whiskers show ±1.5*IQR and center line shows median of 2–3 biological replicates ( e , f ). Data are representative of 2–3 biological replicates ( g ), mean ± s.e.m. of 2–3 biological replicates ( h ), or 6 biological replicates ( i ). P -values by χ 2 test ( a , b ), Fisher’s exact test ( d ), two-tailed Student’s t -tests with Welch’s correction ( e , i ), two-tailed Wilcoxon rank-sum tests ( f ), and ANOVA with Dunnett’s multiple comparison test to E6.5 ( h ). χ 2 = 19.78 ( a ), 85.19 ( b , top), 147.8 ( b , bottom).
    Figure Legend Snippet: Usp9x -mutant embryos arrest at E9.5–E11.5 and display defective repression of early lineage programs marked by H3K27me3. a Genetic cross to delete Usp9x in epiblast derivatives of postimplantation embryos. Quantification of recovered (live) male embryos at several postimplantation stages (right). b Sample images and quantification of control and mutant embryo phenotypes. Relative to controls (left), E9.5 embryos show variable developmental delay, with closed arrow indicating an open anterior neuropore. E11.5 embryos show a range of phenotypes, from hemorrhage to severe delay and death (tally includes dead embryos). Open arrow indicates pericardial edema. Scale bars = 250 µm (E9.5), 2.8 mm (E11.5), with N indicated. c MA plots of expression changes by RNA-seq in two litters of Usp9x mutants versus controls (at E8.5). 3 mutants and 3 controls were sequenced per litter ( N = 12 embryos total; see Supplementary Fig. 3b-d ). d Enrichr analysis of the top-enriched transcription factors (TF) that bind to the genes upregulated in Usp9x -mutant embryos in various cell types. e Expression of the 71 genes upregulated in Usp9x mutants during wild-type development 56 . FC , Fold-change relative to E6.5 embryos. f Distribution and boxplot quantification of H3K27me3 levels 59 over the promoters of genes upregulated in Usp9x mutants (10 kb upstream, 1 kb downstream of TSS). g Representative genome browser tracks of H3K27me3 in wild-type embryos (E6.5–E8.5) at the Nodal locus 59 . Known enhancer elements are highlighted and show gains of H3K27me3. h Nodal mRNA expression in wild-type development 56 . i Nodal mRNA expression in E8.5 Usp9x- mutant or control embryos. Boxplot hinges show the first and third quartiles, whiskers show ±1.5*IQR and center line shows median of 2–3 biological replicates ( e , f ). Data are representative of 2–3 biological replicates ( g ), mean ± s.e.m. of 2–3 biological replicates ( h ), or 6 biological replicates ( i ). P -values by χ 2 test ( a , b ), Fisher’s exact test ( d ), two-tailed Student’s t -tests with Welch’s correction ( e , i ), two-tailed Wilcoxon rank-sum tests ( f ), and ANOVA with Dunnett’s multiple comparison test to E6.5 ( h ). χ 2 = 19.78 ( a ), 85.19 ( b , top), 147.8 ( b , bottom).

    Techniques Used: Mutagenesis, Expressing, RNA Sequencing Assay, Two Tailed Test

    Usp9x promotes ES cell self-renewal and a transcriptional signature of preimplantation linked to PRC2 activity. a Schematic of an auxin-inducible degron (AID) system for acute Usp9x depletion in mouse embryonic stem (ES) cells with representative flow cytometry plot of GFP (AID-Usp9x) expression in Usp9x-low and Usp9x-high ES cells. Right: western blot of endogenous Usp9x level in sorted cell fractions (see Supplementary Fig. 1b ). b Quantification and representative images of colony formation assays. Usp9x-low ES cells display a self-renewal deficit. AP , Alkaline Phosphatase. c Principal Component (PC) Analysis of gene expression by RNA-seq. 8 h : 8 h auxin. No auxin : AID-Usp9x cells with vehicle treatment. 48 h : 8 h auxin followed by 48 h recovery without auxin. Flag : Flag-Usp9x cells after 8 h auxin and 48 h recovery. d The transcriptional signatures of Usp9x-high or Usp9x-low ES cells correlate with different stages of peri-implantation development by Gene Set Enrichment Analysis (GSEA). Genes differentially expressed between Usp9x-high or Usp9x-low ES cells and controls were used in each case. See Methods for references. DE, differentially expressed (relative to controls); NS, not significant (FDR > 0.05); NES, Normalized Enrichment Score. e Usp9x mRNA expression in the epiblast declines from pre- to postimplantation 36 , 37 . f Flow cytometry plot measuring median fluorescence intensity of GFP (Usp9x expression) in Usp9x-high and Usp9x-low ES cells after 8 h auxin treatment and 48 h recovery (without auxin). g Fold-change in expression of all genes at 48 h relative to control cells, showing hypotranscription in Usp9x-high ES cells and hypertranscription in Usp9x-low ES cells. h Heatmaps with summary profile plot of Suz12 binding (data from wild-type ES cells 43 ) over the genes upregulated in Usp9x-low cells or a random subset ( N = 1310). i Boxplots showing repression (in Usp9x-high) or induction (in Usp9x-low) of Suz12 target genes 57 , compared to a random subset ( N = 3350). Western blots represent at least two biological replicates ( a ). Data are mean ± s.d. of four replicates from two independent experiments ( b ), mean ± s.d. of 3–4 replicates ( e ), representative of three experiments ( f , h ). Boxplot hinges ( g , i ) show the first and third quartiles, whiskers show ±1.5*inter-quartile range (IQR) and center line shows median of three biological replicates. **** P
    Figure Legend Snippet: Usp9x promotes ES cell self-renewal and a transcriptional signature of preimplantation linked to PRC2 activity. a Schematic of an auxin-inducible degron (AID) system for acute Usp9x depletion in mouse embryonic stem (ES) cells with representative flow cytometry plot of GFP (AID-Usp9x) expression in Usp9x-low and Usp9x-high ES cells. Right: western blot of endogenous Usp9x level in sorted cell fractions (see Supplementary Fig. 1b ). b Quantification and representative images of colony formation assays. Usp9x-low ES cells display a self-renewal deficit. AP , Alkaline Phosphatase. c Principal Component (PC) Analysis of gene expression by RNA-seq. 8 h : 8 h auxin. No auxin : AID-Usp9x cells with vehicle treatment. 48 h : 8 h auxin followed by 48 h recovery without auxin. Flag : Flag-Usp9x cells after 8 h auxin and 48 h recovery. d The transcriptional signatures of Usp9x-high or Usp9x-low ES cells correlate with different stages of peri-implantation development by Gene Set Enrichment Analysis (GSEA). Genes differentially expressed between Usp9x-high or Usp9x-low ES cells and controls were used in each case. See Methods for references. DE, differentially expressed (relative to controls); NS, not significant (FDR > 0.05); NES, Normalized Enrichment Score. e Usp9x mRNA expression in the epiblast declines from pre- to postimplantation 36 , 37 . f Flow cytometry plot measuring median fluorescence intensity of GFP (Usp9x expression) in Usp9x-high and Usp9x-low ES cells after 8 h auxin treatment and 48 h recovery (without auxin). g Fold-change in expression of all genes at 48 h relative to control cells, showing hypotranscription in Usp9x-high ES cells and hypertranscription in Usp9x-low ES cells. h Heatmaps with summary profile plot of Suz12 binding (data from wild-type ES cells 43 ) over the genes upregulated in Usp9x-low cells or a random subset ( N = 1310). i Boxplots showing repression (in Usp9x-high) or induction (in Usp9x-low) of Suz12 target genes 57 , compared to a random subset ( N = 3350). Western blots represent at least two biological replicates ( a ). Data are mean ± s.d. of four replicates from two independent experiments ( b ), mean ± s.d. of 3–4 replicates ( e ), representative of three experiments ( f , h ). Boxplot hinges ( g , i ) show the first and third quartiles, whiskers show ±1.5*inter-quartile range (IQR) and center line shows median of three biological replicates. **** P

    Techniques Used: Activity Assay, Flow Cytometry, Expressing, Western Blot, RNA Sequencing Assay, Fluorescence, Binding Assay

    22) Product Images from "Global mRNA selection mechanisms for translation initiation"

    Article Title: Global mRNA selection mechanisms for translation initiation

    Journal: Genome Biology

    doi: 10.1186/s13059-014-0559-z

    Caf20p self-regulates its own transcript. (A) Transcripts overrepresented in the eIF4E pull-downs relative to either eIF4G1 (light blue) or eIF4G2 (pink). The data are taken from the GLM model presented in Figure 3 B. (B) A three-dimensional surface plot of P -values detailing the level of significance for the enrichment of the individual closed loop/eIF4E-BP transcripts across the six RIP-seq experiments. (C) A semi-quantitative RT-PCR validation of Caf20p protein’s association with its own transcript using primers designed to the regions 1 to 6 depicted in the figure (detailed in Additional file 6 ). The level from these regions of the CAF20 transcript was determined in TAP affinity purified samples from the CAF20-TAP strains relative to wild-type (WT) strains. (D) A diagram depicting two possible models by which Caf20p could interact with its own transcript to regulate protein production. (E) TAP affinity purification and western blot analysis from eIF4E-TAP tagged strains, investigating the association of eIF4E with both endogenous Caf20p protein and Flag-tagged wild-type Caf20p or Flag-tagged Caf20 m2 p (which has had the eIF4E binding region mutated). (F) Validation of the specificity of RT-PCR primers using total RNA from the strains depicted under the bar chart for either endogenous CAF20 transcripts or Flag-tagged CAF20 transcripts (Additional file 6 ). Error bars are ± standard error from three replicate experiments. (G) qRT-PCR for the endogenous and Flag-tagged CAF20 transcripts from an eIF4E-TAP affinity purification using the primers validated above. The CAF20 or CAF20-fl transcripts are quantified in the IP samples relative to total RNA for the strains listed. Error bars are ± standard error from three replicate experiments. (H) Western blot analysis using extracts from caf20 deletion strains transformed with either centromeric (low copy) plasmids bearing either wild-type CAF20-fl gene or the m2 mutant of CAF20-fl . Three different single transformants are analyzed for each strain and the blots are probed with anti-Flag antibodies to detect Caf20-fl relative to control anti-eIF4A antibodies.
    Figure Legend Snippet: Caf20p self-regulates its own transcript. (A) Transcripts overrepresented in the eIF4E pull-downs relative to either eIF4G1 (light blue) or eIF4G2 (pink). The data are taken from the GLM model presented in Figure 3 B. (B) A three-dimensional surface plot of P -values detailing the level of significance for the enrichment of the individual closed loop/eIF4E-BP transcripts across the six RIP-seq experiments. (C) A semi-quantitative RT-PCR validation of Caf20p protein’s association with its own transcript using primers designed to the regions 1 to 6 depicted in the figure (detailed in Additional file 6 ). The level from these regions of the CAF20 transcript was determined in TAP affinity purified samples from the CAF20-TAP strains relative to wild-type (WT) strains. (D) A diagram depicting two possible models by which Caf20p could interact with its own transcript to regulate protein production. (E) TAP affinity purification and western blot analysis from eIF4E-TAP tagged strains, investigating the association of eIF4E with both endogenous Caf20p protein and Flag-tagged wild-type Caf20p or Flag-tagged Caf20 m2 p (which has had the eIF4E binding region mutated). (F) Validation of the specificity of RT-PCR primers using total RNA from the strains depicted under the bar chart for either endogenous CAF20 transcripts or Flag-tagged CAF20 transcripts (Additional file 6 ). Error bars are ± standard error from three replicate experiments. (G) qRT-PCR for the endogenous and Flag-tagged CAF20 transcripts from an eIF4E-TAP affinity purification using the primers validated above. The CAF20 or CAF20-fl transcripts are quantified in the IP samples relative to total RNA for the strains listed. Error bars are ± standard error from three replicate experiments. (H) Western blot analysis using extracts from caf20 deletion strains transformed with either centromeric (low copy) plasmids bearing either wild-type CAF20-fl gene or the m2 mutant of CAF20-fl . Three different single transformants are analyzed for each strain and the blots are probed with anti-Flag antibodies to detect Caf20-fl relative to control anti-eIF4A antibodies.

    Techniques Used: Quantitative RT-PCR, Affinity Purification, Western Blot, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Transformation Assay, Mutagenesis

    Validation of transcript clusters by quantitative RT-PCR. Figure shows four plots, one for each of the transcript clusters. The indicated mRNAs are quantified in the IP samples relative to total RNA for the untagged control and closed loop/4E-BP regulatory components. Error bars are ± standard error from three replicate experiments.
    Figure Legend Snippet: Validation of transcript clusters by quantitative RT-PCR. Figure shows four plots, one for each of the transcript clusters. The indicated mRNAs are quantified in the IP samples relative to total RNA for the untagged control and closed loop/4E-BP regulatory components. Error bars are ± standard error from three replicate experiments.

    Techniques Used: Quantitative RT-PCR

    23) Product Images from "SAF-A Regulates Interphase Chromosome Structure through Oligomerization with Chromatin-Associated RNAs"

    Article Title: SAF-A Regulates Interphase Chromosome Structure through Oligomerization with Chromatin-Associated RNAs

    Journal: Cell

    doi: 10.1016/j.cell.2017.05.029

    ATP- and RNA-Dependent SAF-A Oligomerization Cycle (A) Left: Western blot for endogenous SAF-A protein extracted from 293T cells treated with or without α-amanitin and stabilized by cross-linking with different concentrations of 1,8-bismaleimido-diethyleneglycol (BM(PEG)2). Proteins were resolved by SDS-PAGE to reveal different SAF-A species (oligomer; D-dimer; M-monomer). Right: Quantification of data in left panel. (B) Left: Western blot for FLAG-tagged SAF-A AAA + /RGG protein expressed in 293T cells and stabilized with different concentrations of disuccinimidyl suberate (DSS). Extracted proteins were resolved by SDS-PAGE. Right: SAF-A wild-type, Walker A, or Walker B mutants expressed in 293T cells, treated with ATP or ATPγS and cross-linked with 0.3 mM BM(PEG)2, extracted, and resolved by SDS-PAGE. (C) 293T cells expressing full-length FLAG-tagged SAF-A pulse-labeled with 5-ethynyl uridine (5-EU) and stabilized with BM(PEG)2. SAF-A was extracted, immuno-purified and resolved by native PAGE. Left: Western blot for SAF-A. Right: RNA detection in SAF-A oligomers. Samples were fractionated by native gel electrophoresis, transferred to membrane, and RNA was labeled by conjugating biotin to pre-incorporated 5-EU using click chemistry and detection using avidin-HRP. (D) Western blot for endogenous SAF-A to analyze protein oligomerization. 293T cells pre-treated with RNaseA, then incubated in the presence or absence of total RNA or apyrase and stabilized by cross-linking with BM(PEG)2. Proteins were extracted and resolved by SDS-PAGE (oligomer; D-dimer; M-monomer). (E) Western blot for FLAG-SAF-A to analyze protein de-oligomerization. Cells were cross-linked with dithio-bis-maleimidoethan (DTME) and stabilized SAF-A was immuno-purified. Cross-links were reversed with DTT, then incubated in the presence of RNase, apyrase, or nucleotides and fractionated by native PAGE. (F) Model for the ATP- and RNA-dependent SAF-A (purple) oligomerization cycle. See also Figure S4 .
    Figure Legend Snippet: ATP- and RNA-Dependent SAF-A Oligomerization Cycle (A) Left: Western blot for endogenous SAF-A protein extracted from 293T cells treated with or without α-amanitin and stabilized by cross-linking with different concentrations of 1,8-bismaleimido-diethyleneglycol (BM(PEG)2). Proteins were resolved by SDS-PAGE to reveal different SAF-A species (oligomer; D-dimer; M-monomer). Right: Quantification of data in left panel. (B) Left: Western blot for FLAG-tagged SAF-A AAA + /RGG protein expressed in 293T cells and stabilized with different concentrations of disuccinimidyl suberate (DSS). Extracted proteins were resolved by SDS-PAGE. Right: SAF-A wild-type, Walker A, or Walker B mutants expressed in 293T cells, treated with ATP or ATPγS and cross-linked with 0.3 mM BM(PEG)2, extracted, and resolved by SDS-PAGE. (C) 293T cells expressing full-length FLAG-tagged SAF-A pulse-labeled with 5-ethynyl uridine (5-EU) and stabilized with BM(PEG)2. SAF-A was extracted, immuno-purified and resolved by native PAGE. Left: Western blot for SAF-A. Right: RNA detection in SAF-A oligomers. Samples were fractionated by native gel electrophoresis, transferred to membrane, and RNA was labeled by conjugating biotin to pre-incorporated 5-EU using click chemistry and detection using avidin-HRP. (D) Western blot for endogenous SAF-A to analyze protein oligomerization. 293T cells pre-treated with RNaseA, then incubated in the presence or absence of total RNA or apyrase and stabilized by cross-linking with BM(PEG)2. Proteins were extracted and resolved by SDS-PAGE (oligomer; D-dimer; M-monomer). (E) Western blot for FLAG-SAF-A to analyze protein de-oligomerization. Cells were cross-linked with dithio-bis-maleimidoethan (DTME) and stabilized SAF-A was immuno-purified. Cross-links were reversed with DTT, then incubated in the presence of RNase, apyrase, or nucleotides and fractionated by native PAGE. (F) Model for the ATP- and RNA-dependent SAF-A (purple) oligomerization cycle. See also Figure S4 .

    Techniques Used: Western Blot, SDS Page, Expressing, Labeling, Purification, Clear Native PAGE, RNA Detection, Nucleic Acid Electrophoresis, Avidin-Biotin Assay, Incubation

    24) Product Images from "RNA-dependent chromatin targeting of TET2 for endogenous retrovirus control in pluripotent stem cells"

    Article Title: RNA-dependent chromatin targeting of TET2 for endogenous retrovirus control in pluripotent stem cells

    Journal: Nature genetics

    doi: 10.1038/s41588-018-0060-9

    PSPC1 and TET2 silence MERVL transcriptionally and post-transcriptionally a , MERVL expression in Tet1/2/3 triple knock-out ( Tet TKO) ESCs rescued with an empty vector (+EV), a wild-type (+TET2WT), or a catalytic mutant (+TET2Mut) TET2. Center line, median; box and whisker plots: ± 10th–90th percentile range. Data are from 5 independent experiments (n=14 total technical replicates for each rescue). Two-tailed Student’s t -test was applied. ns, not significant. b–c , MERVL and IAP enrichment, compared to U6 negative control, among anti-5hmC immunoprecipitated RNAs in Tet TKO (b) and Pspc1 KO (c) ESCs rescued with an empty vector (+EV), a wild-type, or a mutant TET2/PSPC1. Data are presented as mean ± s.e.m. (n=3 independent experiments). Two-tailed Student’s t -test was applied. ns, not significant. d , (Top) Schematic of the protocol used for inhibition of transcription with α-Amanitin for RNA stability assay. (Bottom) Relative abundance of MERVL RNA in Pspc1 WT an d KO ESCs after transcriptional inhibition for 1, 2, or 4 hours with α-Amanitin. Data are normalized to untreated cells at time 0 h (Vehicle without treatment). Error bars indicate s.e.m. (n=3). Two-tailed Student’s t -test was applied. ns, not significant. e , A model of MERVL regulation by PSPC1/TET2 and HDAC1/2 in ESCs. PSPC1 binding to actively transcribed MERVL RNAs recruits TET2 and HDAC1/2 to chromatin. TET2 catalyzes 5hmC modification of MERVL RNAs resulting in their destabilization, and HDAC1/2 deacetylate histones at the chromatin level leading to transcriptional repression of the MERVL loci. Transcriptional and posttranscriptional repression of MERVL leads to the release of the PSPC1-TET2-HDAC1/2 complex from chromatin. Sporadic reactivation of MERVL , via a yet-to-be defined mechanism, leads to the recruitment PSPC1-TET2-HDAC1/2 for transcriptional and posttranscriptional control of MERVL and coordinated gene expression. Illustration by Jill Gregory. Printed with permission of ©Mount Sinai Health System.
    Figure Legend Snippet: PSPC1 and TET2 silence MERVL transcriptionally and post-transcriptionally a , MERVL expression in Tet1/2/3 triple knock-out ( Tet TKO) ESCs rescued with an empty vector (+EV), a wild-type (+TET2WT), or a catalytic mutant (+TET2Mut) TET2. Center line, median; box and whisker plots: ± 10th–90th percentile range. Data are from 5 independent experiments (n=14 total technical replicates for each rescue). Two-tailed Student’s t -test was applied. ns, not significant. b–c , MERVL and IAP enrichment, compared to U6 negative control, among anti-5hmC immunoprecipitated RNAs in Tet TKO (b) and Pspc1 KO (c) ESCs rescued with an empty vector (+EV), a wild-type, or a mutant TET2/PSPC1. Data are presented as mean ± s.e.m. (n=3 independent experiments). Two-tailed Student’s t -test was applied. ns, not significant. d , (Top) Schematic of the protocol used for inhibition of transcription with α-Amanitin for RNA stability assay. (Bottom) Relative abundance of MERVL RNA in Pspc1 WT an d KO ESCs after transcriptional inhibition for 1, 2, or 4 hours with α-Amanitin. Data are normalized to untreated cells at time 0 h (Vehicle without treatment). Error bars indicate s.e.m. (n=3). Two-tailed Student’s t -test was applied. ns, not significant. e , A model of MERVL regulation by PSPC1/TET2 and HDAC1/2 in ESCs. PSPC1 binding to actively transcribed MERVL RNAs recruits TET2 and HDAC1/2 to chromatin. TET2 catalyzes 5hmC modification of MERVL RNAs resulting in their destabilization, and HDAC1/2 deacetylate histones at the chromatin level leading to transcriptional repression of the MERVL loci. Transcriptional and posttranscriptional repression of MERVL leads to the release of the PSPC1-TET2-HDAC1/2 complex from chromatin. Sporadic reactivation of MERVL , via a yet-to-be defined mechanism, leads to the recruitment PSPC1-TET2-HDAC1/2 for transcriptional and posttranscriptional control of MERVL and coordinated gene expression. Illustration by Jill Gregory. Printed with permission of ©Mount Sinai Health System.

    Techniques Used: Expressing, Knock-Out, Plasmid Preparation, Mutagenesis, Whisker Assay, Two Tailed Test, Negative Control, Immunoprecipitation, Inhibition, Stability Assay, Binding Assay, Modification

    25) Product Images from "Efficient Cellular Release of Rift Valley Fever Virus Requires Genomic RNA"

    Article Title: Efficient Cellular Release of Rift Valley Fever Virus Requires Genomic RNA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018070

    Genomic RNA is packaged into RVF-VLPs that lack RdRp. BSR-T7/5 cells were transfected with genome and all of the structural proteins (WT), or one or more of the components was replaced with an equivalent amount of empty vector (-Gn/Gc, -N, -RdRp, -genome, -Gn and -Gc) or with plasmids expressing mutant alleles of Gn or Gc (GnK48 or GcW1). At 24 h the media was replaced with fresh media containing benzonase nuclease. At 48 h the media was removed, clarified and the RVF-VLPs were harvested by ultracentrifugation. The RNA was isolated from the RVF-VLPs and cDNA was generated with primers that recognize the genomic termini and reverse transcriptase (+RT). Duplicate samples were also run without reverse transcriptase (-RT). PCR was performed using primers that flank the intergenic region. The numbers on the left of the gel image indicate size standards.
    Figure Legend Snippet: Genomic RNA is packaged into RVF-VLPs that lack RdRp. BSR-T7/5 cells were transfected with genome and all of the structural proteins (WT), or one or more of the components was replaced with an equivalent amount of empty vector (-Gn/Gc, -N, -RdRp, -genome, -Gn and -Gc) or with plasmids expressing mutant alleles of Gn or Gc (GnK48 or GcW1). At 24 h the media was replaced with fresh media containing benzonase nuclease. At 48 h the media was removed, clarified and the RVF-VLPs were harvested by ultracentrifugation. The RNA was isolated from the RVF-VLPs and cDNA was generated with primers that recognize the genomic termini and reverse transcriptase (+RT). Duplicate samples were also run without reverse transcriptase (-RT). PCR was performed using primers that flank the intergenic region. The numbers on the left of the gel image indicate size standards.

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Mutagenesis, Isolation, Generated, Reverse Transcription Polymerase Chain Reaction

    26) Product Images from "Two Piwi proteins, Xiwi and Xili, are expressed in the Xenopus female germline"

    Article Title: Two Piwi proteins, Xiwi and Xili, are expressed in the Xenopus female germline

    Journal: RNA

    doi: 10.1261/rna.1422509

    Xiwi1 interacts specifically with RNAs ∼30 nt in length. RNAs immunoprecipitated with an antibody directed against Xiwi1 and the corresponding preimmune serum and total small RNA were extracted and 32 P-end-labeled. A specific fraction migrating
    Figure Legend Snippet: Xiwi1 interacts specifically with RNAs ∼30 nt in length. RNAs immunoprecipitated with an antibody directed against Xiwi1 and the corresponding preimmune serum and total small RNA were extracted and 32 P-end-labeled. A specific fraction migrating

    Techniques Used: Immunoprecipitation, Labeling

    27) Product Images from "Novel RNA viruses within plant parasitic cyst nematodes"

    Article Title: Novel RNA viruses within plant parasitic cyst nematodes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0193881

    Characterization of soybean cyst nematode (SCN) bunya-like virus (BLV). ( A ) RNA-dependent RNA polymerase (RdRP; L) of SCN BLV. An identified InterProScan region is shown below the ORF. The scale denotes nucleotide length. ( B ) Phylogenetic tree of SCN BLV RdRP in relation to polymerases of closely related viruses via NCBI PSI-BLAST. Proteins were aligned with ClustalW and trees constructed with Geneious Tree Builder (Jukes-Cantor genetic distance model; neighbor-joining tree method; no outgroup; 1000 replicates; 50% support threshold). Branch labels display consensus support (%).
    Figure Legend Snippet: Characterization of soybean cyst nematode (SCN) bunya-like virus (BLV). ( A ) RNA-dependent RNA polymerase (RdRP; L) of SCN BLV. An identified InterProScan region is shown below the ORF. The scale denotes nucleotide length. ( B ) Phylogenetic tree of SCN BLV RdRP in relation to polymerases of closely related viruses via NCBI PSI-BLAST. Proteins were aligned with ClustalW and trees constructed with Geneious Tree Builder (Jukes-Cantor genetic distance model; neighbor-joining tree method; no outgroup; 1000 replicates; 50% support threshold). Branch labels display consensus support (%).

    Techniques Used: Construct

    28) Product Images from "3′READS+, a sensitive and accurate method for 3′ end sequencing of polyadenylated RNA"

    Article Title: 3′READS+, a sensitive and accurate method for 3′ end sequencing of polyadenylated RNA

    Journal: RNA

    doi: 10.1261/rna.057075.116

    Optimization of 5′ and 3′ adapter ligation steps. ( A ) Ligation protocols tested. In protocol A, ligation with 3′ and 5′ adapters was performed sequentially in the same tube. The 5′ adapter is an RNA oligo with hydroxyl
    Figure Legend Snippet: Optimization of 5′ and 3′ adapter ligation steps. ( A ) Ligation protocols tested. In protocol A, ligation with 3′ and 5′ adapters was performed sequentially in the same tube. The 5′ adapter is an RNA oligo with hydroxyl

    Techniques Used: Ligation

    Digestion of poly(A) RNA with a LNA oligo. ( A , top ) Schematic showing digestion of the poly(A) tail annealed to the T 35 U 15 oligo by RNase H. In theory, the A's hybridized to T's are digested by RNase H, whereas those to U's are not. RNase H digestion
    Figure Legend Snippet: Digestion of poly(A) RNA with a LNA oligo. ( A , top ) Schematic showing digestion of the poly(A) tail annealed to the T 35 U 15 oligo by RNase H. In theory, the A's hybridized to T's are digested by RNase H, whereas those to U's are not. RNase H digestion

    Techniques Used:

    29) Product Images from "DNA Damage, Homology-Directed Repair, and DNA Methylation"

    Article Title: DNA Damage, Homology-Directed Repair, and DNA Methylation

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.0030110

    Mapping of GFP Transcription in Recombinant and Nonrecombinant Cells with and without 5-AzadC Treatment (A) A schematic of the DR-GFP transcriptional unit shows the location of the CMV promoter, intron, and GFP coding sequence. Primers used for quantitative RT-PCR and RT-PCR are indicated by arrows. Different sets of primers were derived from the intron (738–757, forward 5′-CGTTACTCCCACAGGTGAGC-3′; 966–948, reverse 5′-CGCCCGTAGCGCTCACAGC-3′), AUG (1,666–1,685, forward 5′-TACAGCTCCTGGGCAACGTG-3′; 1,911–1,892, reverse, 5′-TCCTGCTCCTGGGCTTCTCG-3′), and BcgI/I-SceI (described in Figure 1 A) segments of GFP gene. Control (DR-GFP cells transfected with pBluescript), HR-L, and HR-H cells were treated with 40 μM 5-AzadC for 48 h. Total RNA was extracted 24 h later and subjected to quantitative RT-PCR with the primers indicated. The data, derived from three independent cDNAs, are shown as fold induction by 5-AzadC over the basal control, normalized to GADPH and β-actin. The primers used to amplify the control samples were those indicated as I-SceI unrec ( Figure 1 A). (B) Shown is RT-PCR with the same cDNAs indicated in (A) at 30 cycles.
    Figure Legend Snippet: Mapping of GFP Transcription in Recombinant and Nonrecombinant Cells with and without 5-AzadC Treatment (A) A schematic of the DR-GFP transcriptional unit shows the location of the CMV promoter, intron, and GFP coding sequence. Primers used for quantitative RT-PCR and RT-PCR are indicated by arrows. Different sets of primers were derived from the intron (738–757, forward 5′-CGTTACTCCCACAGGTGAGC-3′; 966–948, reverse 5′-CGCCCGTAGCGCTCACAGC-3′), AUG (1,666–1,685, forward 5′-TACAGCTCCTGGGCAACGTG-3′; 1,911–1,892, reverse, 5′-TCCTGCTCCTGGGCTTCTCG-3′), and BcgI/I-SceI (described in Figure 1 A) segments of GFP gene. Control (DR-GFP cells transfected with pBluescript), HR-L, and HR-H cells were treated with 40 μM 5-AzadC for 48 h. Total RNA was extracted 24 h later and subjected to quantitative RT-PCR with the primers indicated. The data, derived from three independent cDNAs, are shown as fold induction by 5-AzadC over the basal control, normalized to GADPH and β-actin. The primers used to amplify the control samples were those indicated as I-SceI unrec ( Figure 1 A). (B) Shown is RT-PCR with the same cDNAs indicated in (A) at 30 cycles.

    Techniques Used: Recombinant, Sequencing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Transfection

    30) Product Images from "Rbfox2 is Critical for Maintaining Alternative Polyadenylation and Mitochondrial Health in Myoblasts"

    Article Title: Rbfox2 is Critical for Maintaining Alternative Polyadenylation and Mitochondrial Health in Myoblasts

    Journal: bioRxiv

    doi: 10.1101/2020.05.13.093013

    RBFOX2 binding sites are enriched upstream of poly(A) sites in 3’UTRs that undergo APA changes. (A) Metagene analysis of RBFOX2 CLIP-binding distribution with respect to PASs in all detectable PASs, or in upregulated/downregulated PASs identified in RBFOX2 KD H9c2 cells. (B) Genome browser image of Rab7a 3’UTR displays tandem-APA changes determined by PAC-seq (left). Tandem-APA change in Rab7a in control vs RBFOX2 KD H9c2 cells determined by RT-qPCR (right). The ratio of “distal or long” to “common” mRNA expression levels of Rab7a by RT-qPCR in control cells was normalized to 1. Statistical significance was calculated using unpaired t-test to compare two different groups in three independent experiments (n=3). Data represent means ± SD. *p=0.0466. (C) RBFOX2 CLIP-seq reads mapped to the Rab7a 3’UTR. (D) FLAG western blot of FLAG-RBFOX2 pulled down with in-vitro transcribed and biotinylated Rab7a transcripts in induced or uninduced HEK293 cells. No RNA was used as a negative control for non-specific protein binding in induced or uninduced cells.
    Figure Legend Snippet: RBFOX2 binding sites are enriched upstream of poly(A) sites in 3’UTRs that undergo APA changes. (A) Metagene analysis of RBFOX2 CLIP-binding distribution with respect to PASs in all detectable PASs, or in upregulated/downregulated PASs identified in RBFOX2 KD H9c2 cells. (B) Genome browser image of Rab7a 3’UTR displays tandem-APA changes determined by PAC-seq (left). Tandem-APA change in Rab7a in control vs RBFOX2 KD H9c2 cells determined by RT-qPCR (right). The ratio of “distal or long” to “common” mRNA expression levels of Rab7a by RT-qPCR in control cells was normalized to 1. Statistical significance was calculated using unpaired t-test to compare two different groups in three independent experiments (n=3). Data represent means ± SD. *p=0.0466. (C) RBFOX2 CLIP-seq reads mapped to the Rab7a 3’UTR. (D) FLAG western blot of FLAG-RBFOX2 pulled down with in-vitro transcribed and biotinylated Rab7a transcripts in induced or uninduced HEK293 cells. No RNA was used as a negative control for non-specific protein binding in induced or uninduced cells.

    Techniques Used: Binding Assay, Cross-linking Immunoprecipitation, Quantitative RT-PCR, Expressing, Western Blot, In Vitro, Negative Control, Protein Binding

    31) Product Images from "Capping-RACE: a simple, accurate, and sensitive 5′ RACE method for use in prokaryotes"

    Article Title: Capping-RACE: a simple, accurate, and sensitive 5′ RACE method for use in prokaryotes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky739

    Experimental verification of Capping-RACE with in vitro transcribed RNA. ( A ) Different treatments of in vitro transcribed RNA. ( B ) The cDNA products from the differently treated RNA described in (A). Lane 1, the negative control, performed without adding reverse transcriptase (RT). Lane 2, in vitro transcribed RNA that was not subjected to any treatment. Lane 3, in vitro transcribed RNA subjected to RppH treatment. Lane 4, in vitro transcribed RNA subjected to dual treatment, i.e. RppH treatment prior to VCE treatment. Lane 5, in vitro transcribed RNA subjected to vaccinia capping enzyme (VCE) treatment. The reaction products were analysed on a 12% non-denaturing polyacrylamide gel and detected by a fluorescence image analyser (FUJIFILM, FLA-5100).
    Figure Legend Snippet: Experimental verification of Capping-RACE with in vitro transcribed RNA. ( A ) Different treatments of in vitro transcribed RNA. ( B ) The cDNA products from the differently treated RNA described in (A). Lane 1, the negative control, performed without adding reverse transcriptase (RT). Lane 2, in vitro transcribed RNA that was not subjected to any treatment. Lane 3, in vitro transcribed RNA subjected to RppH treatment. Lane 4, in vitro transcribed RNA subjected to dual treatment, i.e. RppH treatment prior to VCE treatment. Lane 5, in vitro transcribed RNA subjected to vaccinia capping enzyme (VCE) treatment. The reaction products were analysed on a 12% non-denaturing polyacrylamide gel and detected by a fluorescence image analyser (FUJIFILM, FLA-5100).

    Techniques Used: In Vitro, Negative Control, Fluorescence

    32) Product Images from "Hibernation factors directly block ribonucleases from entering the ribosome in response to starvation"

    Article Title: Hibernation factors directly block ribonucleases from entering the ribosome in response to starvation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab017

    YbeY and RNase R are involved in generation and removal of accumulating fragments. ( A ) Schematic of 16S rRNA and DNA oligonucleotide probes used for Northern blot analysis in (B) and (C). For the sequences of the indicated probes see Supplementary Table S2 . ( B ) Northern blot analysis of 16S rRNA fragments found in total RNA of hibernation factor mutants and RNase mutants after five days of starvation. The schematics on the right show detected fragments. Grey bars indicate fragments common to both WT and ΔHF, red bars indicate fragments only present in ΔHF. Pink bars indicate overhang in fragments I* and V* of Δ rnr and Δ ybeY mutants. For additional probes and magnified selected areas in the range of fragment I and fragment V see Supplementary Figure S9 . ( C ) As described in (B). RNA was resolved on 6% urea–PAGE gels for detection of RNA in a lower size range. The schematic on the right shows fragments VI and VII mapped by urea–PAGE and Northern blot analysis. For additional probes see Supplementary Figure S9 .
    Figure Legend Snippet: YbeY and RNase R are involved in generation and removal of accumulating fragments. ( A ) Schematic of 16S rRNA and DNA oligonucleotide probes used for Northern blot analysis in (B) and (C). For the sequences of the indicated probes see Supplementary Table S2 . ( B ) Northern blot analysis of 16S rRNA fragments found in total RNA of hibernation factor mutants and RNase mutants after five days of starvation. The schematics on the right show detected fragments. Grey bars indicate fragments common to both WT and ΔHF, red bars indicate fragments only present in ΔHF. Pink bars indicate overhang in fragments I* and V* of Δ rnr and Δ ybeY mutants. For additional probes and magnified selected areas in the range of fragment I and fragment V see Supplementary Figure S9 . ( C ) As described in (B). RNA was resolved on 6% urea–PAGE gels for detection of RNA in a lower size range. The schematic on the right shows fragments VI and VII mapped by urea–PAGE and Northern blot analysis. For additional probes see Supplementary Figure S9 .

    Techniques Used: Northern Blot, Polyacrylamide Gel Electrophoresis

    33) Product Images from "TP53 drives abscopal effect by secretion of senescence-associated molecular signals in non-small cell lung cancer"

    Article Title: TP53 drives abscopal effect by secretion of senescence-associated molecular signals in non-small cell lung cancer

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-021-01883-0

    EVs secreted by high-dose irradiated A549 cells induce in vitro abscopal effects. a EVs secreted by irradiated A549 stimulate the synthesis of DNA:RNA hybrid structures. Immunofluorescence staining with S9.6 antibody. The images are representative of unIR A549 cells exposed to EVs isolated from culture medium of A549 cells non irradiated or irradiated at different doses. Cell images were captured by Nikon Eclipse Ti2 confocal microscope with 60x plan apochromat oil immersion objective lens. Data are presented as grand median ± MAD. b EVs secreted by irradiated A549 induce senescent phenotype. (I) Representative images of SA-β-gal staining of UnIR A549 cells after exposure to EVs isolated from culture medium of IR A549 cells. II. Colony-forming assay (CFA) . The cells were exposed to EVs isolated from UnIR or IR A549 cells for 14 days. 0.05% crystal violet solution was used to visualize the generated colonies with more than 50 cells, which were quantified under inverted microscope (Olympus IX51 microscope, Olympus Corporation, Tokyo, Japan) by two independent observers. III. Expression levels of senescence markers. p21, IL6 mRNA levels were measured by Real-Time PCR and normalized to GAPDH and HPRT-1. Data are the mean of two independent experiments. (C) EVs secreted by irradiated A549 induce M1 polarization in a murine macrophage cell line. Total RNA from RAW 264.7 M0 cells (see Methods section) exposed to EVs secreted by unirradiated, 10 Gy or 20 Gy-irradiated A549 cells were analyzed by RT-PCR for the expression of representative murine M2 genes (Arg1, Egr2) and M1/pro-inflammatory cytokines (IL-6, IL-1β). Expression data are given as fold increase over the mRNA level expressed by RAW 264.7 M0 exposed to UnIR EVs. Data are represented as mean ± SD of triplicate values of two independent experiments (* p
    Figure Legend Snippet: EVs secreted by high-dose irradiated A549 cells induce in vitro abscopal effects. a EVs secreted by irradiated A549 stimulate the synthesis of DNA:RNA hybrid structures. Immunofluorescence staining with S9.6 antibody. The images are representative of unIR A549 cells exposed to EVs isolated from culture medium of A549 cells non irradiated or irradiated at different doses. Cell images were captured by Nikon Eclipse Ti2 confocal microscope with 60x plan apochromat oil immersion objective lens. Data are presented as grand median ± MAD. b EVs secreted by irradiated A549 induce senescent phenotype. (I) Representative images of SA-β-gal staining of UnIR A549 cells after exposure to EVs isolated from culture medium of IR A549 cells. II. Colony-forming assay (CFA) . The cells were exposed to EVs isolated from UnIR or IR A549 cells for 14 days. 0.05% crystal violet solution was used to visualize the generated colonies with more than 50 cells, which were quantified under inverted microscope (Olympus IX51 microscope, Olympus Corporation, Tokyo, Japan) by two independent observers. III. Expression levels of senescence markers. p21, IL6 mRNA levels were measured by Real-Time PCR and normalized to GAPDH and HPRT-1. Data are the mean of two independent experiments. (C) EVs secreted by irradiated A549 induce M1 polarization in a murine macrophage cell line. Total RNA from RAW 264.7 M0 cells (see Methods section) exposed to EVs secreted by unirradiated, 10 Gy or 20 Gy-irradiated A549 cells were analyzed by RT-PCR for the expression of representative murine M2 genes (Arg1, Egr2) and M1/pro-inflammatory cytokines (IL-6, IL-1β). Expression data are given as fold increase over the mRNA level expressed by RAW 264.7 M0 exposed to UnIR EVs. Data are represented as mean ± SD of triplicate values of two independent experiments (* p

    Techniques Used: Irradiation, In Vitro, Immunofluorescence, Staining, Isolation, Microscopy, Generated, Inverted Microscopy, Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    34) Product Images from "Optimized design of antisense oligomers for targeted rRNA depletion"

    Article Title: Optimized design of antisense oligomers for targeted rRNA depletion

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa1072

    ( A ) Genome browser track showing read coverage over X. laevis 12S and 16S rDNA, illustrating the effect of rRNA depletion using variably spaced oligos (bottom). The two largest untiled regions are highlighted. ( B ) Biplot showing sequencing read depth at the center of each untiled region (gap) over 12S and 16S as a function of length of the region. ( C ) Genome browser tracks showing read coverage for hist1h2bj.L, not targeted for depletion; COX2, targeted using oligos with high melting temperature ( T m ); and COX3, targeted using oligos with low T m . Depletion reactions at 45°C for 30 min and 65°C for 5 min are compared to a reaction lacking the gene-targeting oligos. ( D ) Barplot for the three genes as in (C) showing the ratio of RNA-seq transcripts per million in depletion reactions (45°C left bars, 65°C right bars) over the non depleted condition. RPM = reads per million.
    Figure Legend Snippet: ( A ) Genome browser track showing read coverage over X. laevis 12S and 16S rDNA, illustrating the effect of rRNA depletion using variably spaced oligos (bottom). The two largest untiled regions are highlighted. ( B ) Biplot showing sequencing read depth at the center of each untiled region (gap) over 12S and 16S as a function of length of the region. ( C ) Genome browser tracks showing read coverage for hist1h2bj.L, not targeted for depletion; COX2, targeted using oligos with high melting temperature ( T m ); and COX3, targeted using oligos with low T m . Depletion reactions at 45°C for 30 min and 65°C for 5 min are compared to a reaction lacking the gene-targeting oligos. ( D ) Barplot for the three genes as in (C) showing the ratio of RNA-seq transcripts per million in depletion reactions (45°C left bars, 65°C right bars) over the non depleted condition. RPM = reads per million.

    Techniques Used: Sequencing, RNA Sequencing Assay

    35) Product Images from "Histone chaperone Nucleophosmin regulates transcription of key genes involved in oral tumorigenesis"

    Article Title: Histone chaperone Nucleophosmin regulates transcription of key genes involved in oral tumorigenesis

    Journal: bioRxiv

    doi: 10.1101/852095

    AcNPM1 co-occupies with RNA Pol II, chromatin remodeling factors and transcription factors at transcriptional regulatory elements. (A) Plot showing the percent number of AcNPM1 peaks overlapped with ChromHMM + Segway combined segmentation for HeLa S3 genome from the UCSC genome browser. (Key: TSS, predicted promoter region including TSS; PF, predicted promoter flanking region; E, enhancer; WE, predicted weak enhancer or open chromatin cis-regulatory element; CTCF, CTCF enriched element; T, predicted transcribed region; R, predicted repressed or low activity region; None, unclassified). (B) Percent number of TSS and enhancer regions identified by ChromHMM + Segway combined segmentation for HeLa S3, overlapped with AcNPM1 peaks. (C) UCSC genome browser snapshot showing AcNPM1 enrichment at TSS and enhancer regions defined by ChromHMM + Segway combined segmentation for HeLa S3 genome. (Key: TSS, predicted promoter region including TSS; E, enhancer). (D) Boxplots showing AcNPM1 read density on AcNPM1 peaks that overlap or do not overlap DNase I hypersensitive sites (DHSs). (E) Boxplots showing AcNPM1 read density on AcNPM1 peaks with high or low enrichment of H3K27ac. (F-G) Boxplots showing AcNPM1 read density on AcNPM1 peaks that overlap or do not overlap (F) p300 and (G) RNA Pol II (Pol2). (H) Transcription factor binding motifs enriched in AcNPM1 peaks and broadly grouped by transcription factor family. P -value
    Figure Legend Snippet: AcNPM1 co-occupies with RNA Pol II, chromatin remodeling factors and transcription factors at transcriptional regulatory elements. (A) Plot showing the percent number of AcNPM1 peaks overlapped with ChromHMM + Segway combined segmentation for HeLa S3 genome from the UCSC genome browser. (Key: TSS, predicted promoter region including TSS; PF, predicted promoter flanking region; E, enhancer; WE, predicted weak enhancer or open chromatin cis-regulatory element; CTCF, CTCF enriched element; T, predicted transcribed region; R, predicted repressed or low activity region; None, unclassified). (B) Percent number of TSS and enhancer regions identified by ChromHMM + Segway combined segmentation for HeLa S3, overlapped with AcNPM1 peaks. (C) UCSC genome browser snapshot showing AcNPM1 enrichment at TSS and enhancer regions defined by ChromHMM + Segway combined segmentation for HeLa S3 genome. (Key: TSS, predicted promoter region including TSS; E, enhancer). (D) Boxplots showing AcNPM1 read density on AcNPM1 peaks that overlap or do not overlap DNase I hypersensitive sites (DHSs). (E) Boxplots showing AcNPM1 read density on AcNPM1 peaks with high or low enrichment of H3K27ac. (F-G) Boxplots showing AcNPM1 read density on AcNPM1 peaks that overlap or do not overlap (F) p300 and (G) RNA Pol II (Pol2). (H) Transcription factor binding motifs enriched in AcNPM1 peaks and broadly grouped by transcription factor family. P -value

    Techniques Used: Activity Assay, Binding Assay

    36) Product Images from "Transposase assisted tagmentation of RNA/DNA hybrid duplexes"

    Article Title: Transposase assisted tagmentation of RNA/DNA hybrid duplexes

    Journal: bioRxiv

    doi: 10.1101/2020.01.29.926105

    Tagmentation activity of Tn5 transposome on RNA/DNA hybrids. (a) Denaturing (8 M urea) polyacrylamide gel analysis of reverse transcription products of an in vitro transcribed mRNA (IRF9). Lane 1: ssRNA marker. Lane 2: in vitro transcribed mRNA (IRF9). Lane 3 4: reverse transcription products of an in vitro transcribed mRNA (IRF9). Lane 5: reverse transcription product treated with DNase I. Lane 6: reverse transcription product treated with RNase H. ssRNA and ssDNA is marked with a red asterisk and a blue pound sign, respectively. (b) Gel picture showing size distribution of RNA/DNA hybrids products of 50 μl reaction systems without Tn5 transposome, and with 5 μl, 10 μl, and 15 μl Tn5 transposome, respectively. The blue and orange patches denote small and large fragments, respectively. (c) qPCR amplification curve of tagmentation products without Tn5 treatment or with Tn5 treatment in three different buffers (see methods). Average Ct values are 26.41, 18.39, 18.33 and 18.34, respectively. (d) Sanger sequencing chromatograms of PCR products following RNA/DNA hybrid tagmentation and strand extension. Adaptor A and B sequences are highlighted with blue background color and insert sequences are highlighted with yellow background.
    Figure Legend Snippet: Tagmentation activity of Tn5 transposome on RNA/DNA hybrids. (a) Denaturing (8 M urea) polyacrylamide gel analysis of reverse transcription products of an in vitro transcribed mRNA (IRF9). Lane 1: ssRNA marker. Lane 2: in vitro transcribed mRNA (IRF9). Lane 3 4: reverse transcription products of an in vitro transcribed mRNA (IRF9). Lane 5: reverse transcription product treated with DNase I. Lane 6: reverse transcription product treated with RNase H. ssRNA and ssDNA is marked with a red asterisk and a blue pound sign, respectively. (b) Gel picture showing size distribution of RNA/DNA hybrids products of 50 μl reaction systems without Tn5 transposome, and with 5 μl, 10 μl, and 15 μl Tn5 transposome, respectively. The blue and orange patches denote small and large fragments, respectively. (c) qPCR amplification curve of tagmentation products without Tn5 treatment or with Tn5 treatment in three different buffers (see methods). Average Ct values are 26.41, 18.39, 18.33 and 18.34, respectively. (d) Sanger sequencing chromatograms of PCR products following RNA/DNA hybrid tagmentation and strand extension. Adaptor A and B sequences are highlighted with blue background color and insert sequences are highlighted with yellow background.

    Techniques Used: Activity Assay, In Vitro, Marker, Real-time Polymerase Chain Reaction, Amplification, Sequencing, Polymerase Chain Reaction

    37) Product Images from "The dinucleotide composition of the Zika virus genome is shaped by conflicting evolutionary pressures in mammalian hosts and mosquito vectors"

    Article Title: The dinucleotide composition of the Zika virus genome is shaped by conflicting evolutionary pressures in mammalian hosts and mosquito vectors

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.3001201

    Fitness comparison between WT and mutant ZIKV in human and mosquito cells. Human A549 cells, A549 ZAP knockout cells, and AP-61 mosquito cells were infected with equal RNA concentrations of WT ZIKV mixed with either the SCR (A) , CpG-high viruses (CpG_1.0 and CpG_max) (B, C), or UpA_max (D) . Virus was passaged to new cells twice, before total RNA was isolated. ZIKV RNA was amplified with RT-PCR and digested with BsaI (A), SalI (B, C), or HeaIII (D) restriction enzymes to distinguish between WT and the respective mutant viruses. DNA gel images display the largest most distinctive products from each digestion. For more information, see S3 Fig . From left to right images display the digested DNA of the indicated individual viruses, followed by 2 independent experimental repeats performed with separately rescued virus populations (numbered 1 and 2). RT-PCR, reverse transcription PCR; SCR, scrambled control virus; WT, wild-type; ZAP, zinc-finger antiviral protein; ZIKV, Zika virus.
    Figure Legend Snippet: Fitness comparison between WT and mutant ZIKV in human and mosquito cells. Human A549 cells, A549 ZAP knockout cells, and AP-61 mosquito cells were infected with equal RNA concentrations of WT ZIKV mixed with either the SCR (A) , CpG-high viruses (CpG_1.0 and CpG_max) (B, C), or UpA_max (D) . Virus was passaged to new cells twice, before total RNA was isolated. ZIKV RNA was amplified with RT-PCR and digested with BsaI (A), SalI (B, C), or HeaIII (D) restriction enzymes to distinguish between WT and the respective mutant viruses. DNA gel images display the largest most distinctive products from each digestion. For more information, see S3 Fig . From left to right images display the digested DNA of the indicated individual viruses, followed by 2 independent experimental repeats performed with separately rescued virus populations (numbered 1 and 2). RT-PCR, reverse transcription PCR; SCR, scrambled control virus; WT, wild-type; ZAP, zinc-finger antiviral protein; ZIKV, Zika virus.

    Techniques Used: Mutagenesis, Knock-Out, Infection, Isolation, Amplification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    38) Product Images from "Novel RNA viruses within plant parasitic cyst nematodes"

    Article Title: Novel RNA viruses within plant parasitic cyst nematodes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0193881

    Characterization of soybean cyst nematode (SCN) bunya-like virus (BLV). ( A ) RNA-dependent RNA polymerase (RdRP; L) of SCN BLV. An identified InterProScan region is shown below the ORF. The scale denotes nucleotide length. ( B ) Phylogenetic tree of SCN BLV RdRP in relation to polymerases of closely related viruses via NCBI PSI-BLAST. Proteins were aligned with ClustalW and trees constructed with Geneious Tree Builder (Jukes-Cantor genetic distance model; neighbor-joining tree method; no outgroup; 1000 replicates; 50% support threshold). Branch labels display consensus support (%).
    Figure Legend Snippet: Characterization of soybean cyst nematode (SCN) bunya-like virus (BLV). ( A ) RNA-dependent RNA polymerase (RdRP; L) of SCN BLV. An identified InterProScan region is shown below the ORF. The scale denotes nucleotide length. ( B ) Phylogenetic tree of SCN BLV RdRP in relation to polymerases of closely related viruses via NCBI PSI-BLAST. Proteins were aligned with ClustalW and trees constructed with Geneious Tree Builder (Jukes-Cantor genetic distance model; neighbor-joining tree method; no outgroup; 1000 replicates; 50% support threshold). Branch labels display consensus support (%).

    Techniques Used: Construct

    39) Product Images from "Assembly, purification and crystallization of an active HIV-1 reverse transcriptase initiation complex"

    Article Title: Assembly, purification and crystallization of an active HIV-1 reverse transcriptase initiation complex

    Journal: Nucleic Acids Research

    doi:

    Denaturing gel showing use of transcribed tRNA Lys3 as a primer for DNA synthesis by HIV-1 RT. A concentration of 5 µM annealed tRNA Lys3 /PBS-K2 RNA was incubated for 15 min at 37°C without (–; lanes 1A, 2A, 3A) or with (+; lanes 1B, 2B, 3B) 5 µM HIV-1 RT in the presence of 1 mM each dNTP. Prior to use in the initiation reaction, the 2′–3′ cyclic phosphate at the 3′ end of tRNA Lys3 , which results from cleavage by the 3′ HH ribozyme, either was not removed (lanes 1A and 1B), was partially removed (lanes 2A and 2B) or was completely removed (lanes 3A and 3B), as described in Materials and Methods. Migration positions of unextended (tRNA) or fully extended (tRNA–DNA) primer, as visualized by ethidium bromide staining, are indicated. The dephosphorylated tRNA is the slower migrating of the two tRNA species.
    Figure Legend Snippet: Denaturing gel showing use of transcribed tRNA Lys3 as a primer for DNA synthesis by HIV-1 RT. A concentration of 5 µM annealed tRNA Lys3 /PBS-K2 RNA was incubated for 15 min at 37°C without (–; lanes 1A, 2A, 3A) or with (+; lanes 1B, 2B, 3B) 5 µM HIV-1 RT in the presence of 1 mM each dNTP. Prior to use in the initiation reaction, the 2′–3′ cyclic phosphate at the 3′ end of tRNA Lys3 , which results from cleavage by the 3′ HH ribozyme, either was not removed (lanes 1A and 1B), was partially removed (lanes 2A and 2B) or was completely removed (lanes 3A and 3B), as described in Materials and Methods. Migration positions of unextended (tRNA) or fully extended (tRNA–DNA) primer, as visualized by ethidium bromide staining, are indicated. The dephosphorylated tRNA is the slower migrating of the two tRNA species.

    Techniques Used: DNA Synthesis, Concentration Assay, Incubation, Migration, Staining

    40) Product Images from "Characterisation of the developing heart in a pressure overloaded model utilising RNA sequencing to direct functional analysis"

    Article Title: Characterisation of the developing heart in a pressure overloaded model utilising RNA sequencing to direct functional analysis

    Journal: Journal of Anatomy

    doi: 10.1111/joa.13112

    OFT‐banded hearts show overall consistency of gene expression with targeted gene analysis showing differential expression at HH29 and HH35. (A) PCA of RNA sequencing FPKM shows consistency of expression within OFT‐banded (OFT) and sham groups, with a distinction seen between groups. (B) Expression analysis of targeted genes at HH29 and HH35 by qPCR shows a general increase in differential expression in OFT‐banded hearts, with the exception of LDHB and ENS‐1, where decreases are seen (* P
    Figure Legend Snippet: OFT‐banded hearts show overall consistency of gene expression with targeted gene analysis showing differential expression at HH29 and HH35. (A) PCA of RNA sequencing FPKM shows consistency of expression within OFT‐banded (OFT) and sham groups, with a distinction seen between groups. (B) Expression analysis of targeted genes at HH29 and HH35 by qPCR shows a general increase in differential expression in OFT‐banded hearts, with the exception of LDHB and ENS‐1, where decreases are seen (* P

    Techniques Used: Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction

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    Article Snippet: .. We sequenced mRNA in bulk from WM989-A6 populations as per Shaffer et. al. We isolated mRNA and built sequencing libraries using the NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext Ultra RNA Library Prep Kit for Illumina. ..

    Article Title: Transcriptomic Analysis of Differentially Expressed Genes during Flower Organ Development in Genetic Male Sterile and Male Fertile Tagetes erecta by Digital Gene-Expression Profiling
    Article Snippet: Library preparation and sequencing RNA Samples were sent to Novogene Bioinformatics Technology Co. Ltd (Beijing), where the libraries were constructed and sequenced using Illumina HiSeq 2000 platform. .. Sequencing libraries were generated using NEBNext Ultra™ RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s protocols and index codes were added to attribute sequences to each sample. ..

    Generated:

    Article Title: Transcriptomic Analysis of Differentially Expressed Genes during Flower Organ Development in Genetic Male Sterile and Male Fertile Tagetes erecta by Digital Gene-Expression Profiling
    Article Snippet: Library preparation and sequencing RNA Samples were sent to Novogene Bioinformatics Technology Co. Ltd (Beijing), where the libraries were constructed and sequenced using Illumina HiSeq 2000 platform. .. Sequencing libraries were generated using NEBNext Ultra™ RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s protocols and index codes were added to attribute sequences to each sample. ..

    Synthesized:

    Article Title: Multicomponent nature underlies the extraordinary mechanical properties of spider dragline silk
    Article Snippet: All cDNA libraries were constructed according to the standard protocol of the NEBNext Ultra RNA Library Prep Kit for Illumina (New England BioLabs). .. The synthesized double-stranded cDNA was end-repaired using NEBNext End Prep Enzyme Mix before ligation with NEBNext Adaptor for Illumina. .. After USER enzyme treatment, cDNA was amplified by PCR with the following conditions: 20 µL cDNA, 2.5 µL Index Primer, 2.5 µL Universal PCR Primer, 25 µL NEBNext Q5 Hot Start HiFi PCR Master Mix 2X; 98 °C for 30 s and 12 cycles each of 98 °C for 10 s, 65 °C for 75 s and 65 °C for 5 min. cDNA sequencing was conducted with a NextSeq 500 instrument (Illumina) using 150-bp paired-end reads with a NextSeq 500 High Output Kit (300 cycles).

    Ligation:

    Article Title: Multicomponent nature underlies the extraordinary mechanical properties of spider dragline silk
    Article Snippet: All cDNA libraries were constructed according to the standard protocol of the NEBNext Ultra RNA Library Prep Kit for Illumina (New England BioLabs). .. The synthesized double-stranded cDNA was end-repaired using NEBNext End Prep Enzyme Mix before ligation with NEBNext Adaptor for Illumina. .. After USER enzyme treatment, cDNA was amplified by PCR with the following conditions: 20 µL cDNA, 2.5 µL Index Primer, 2.5 µL Universal PCR Primer, 25 µL NEBNext Q5 Hot Start HiFi PCR Master Mix 2X; 98 °C for 30 s and 12 cycles each of 98 °C for 10 s, 65 °C for 75 s and 65 °C for 5 min. cDNA sequencing was conducted with a NextSeq 500 instrument (Illumina) using 150-bp paired-end reads with a NextSeq 500 High Output Kit (300 cycles).

    Construct:

    Article Title: Distinct Transcriptional and Alternative Splicing Signatures of Decidual CD4+ T Cells in Early Human Pregnancy
    Article Snippet: .. We used 200 ng of RNA per sample (three samples of both pCD4 and dCD4 T cells) as input material for the RNA sample preparations. cDNA libraries were constructed using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA), according to the manufacturer’s instructions. .. The products were purified using the AMPure XP system and library quality was determined on the Agilent Bioanalyzer 2100 system.

    Plasmid Preparation:

    Article Title: SDG8-Mediated Histone Methylation and RNA Processing Function in the Response to Nitrate Signaling
    Article Snippet: Protoplasts isolated from shoots were treated with 20 m m of KNO3 and 20 m m of NH4 NO3 before the nuclear import of transcription factor induced by dexamethasone. .. Cells overexpressing either CCT101 isoforms or empty vector controls were collected in triplicate in the same batch of experiment and RNA-Seq libraries were prepared from mRNA using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs). .. All three isoforms are stably expressed at comparable levels based on RNA-Seq readout.

    RNA Sequencing Assay:

    Article Title: SDG8-Mediated Histone Methylation and RNA Processing Function in the Response to Nitrate Signaling
    Article Snippet: Protoplasts isolated from shoots were treated with 20 m m of KNO3 and 20 m m of NH4 NO3 before the nuclear import of transcription factor induced by dexamethasone. .. Cells overexpressing either CCT101 isoforms or empty vector controls were collected in triplicate in the same batch of experiment and RNA-Seq libraries were prepared from mRNA using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs). .. All three isoforms are stably expressed at comparable levels based on RNA-Seq readout.

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    New England Biolabs rna
    <t>RNA</t> isolation and RT-qPCR in microgravity. (A) Photo of SPM. (B) Typical RNA quality from SPM with E . coli (left panel) and mouse liver (right panel), control Qiagen (left lane) SPM (right lane). Center panel shows RNA quality from the 1 g control (left lane) and the returned microgravity sample from ISS (right lane). (C-E) Scatter plots with jitter of the microgravity and 1 g control E . coli singleplex (C), duplex (D) and triplex (E) reactions. One outlier is indicated by the open marker in C. One of the microgravity triplex tubes did not give a dnaK-FAM signal (E). (F-H) Scatter plots with jitter of the microgravity and 1 g control mouse liver singleplex (F), duplex (G) and triplex (H) reactions. One outlier from the microgravity triplex <t>fn1</t> plot is indicated by the open marker and no gapdh-FAM signal was seen in the microgravity triplex reactions (H).
    Rna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs bacteriophage sp6 rna polymerase
    The frameshift reporter construct pKA1. (a) Plasmid pKA1 was derived from pFScass 6 (Brierley et al ., 1992) by site-directed mutagenesis (see Materials and Methods). pKA1 contains a truncated version of the minimal IBV pseudoknot (white box) cloned into a reporter gene, the influenza PB2 gene (shaded boxes). Linearisation of the plasmid with Bam H1 and in vitro transcription using <t>SP6</t> <t>RNA</t> polymerase yields an mRNA (2.4kb) that, when translated in RRL, is predicted to produce a 19 kDa non-frameshift product corresponding to ribosomes that terminate at the UGA termination codon (located immediately downstream of the slippery sequence UUUAAAC, shaded), and a 22 kDa −1 frameshift product. The 0-frame and −2/+1 frames are also open (to some extent) in this construct. Ribosomes which enter these frames produce 28 kDa and 85 kDa products respectively. A bacteriophage T7 promoter is present just upstream of the frameshift region; this promoter is employed to generate short, pseudoknot-containing transcripts from Bst NI-digested templates for secondary structure analysis. (b) The wild-type (wt) IBV, the minimal IBV and the pKA1 pseudoknots (PK). The minimal IBV frameshift signal present in pFScass 6 is based on the wild-type IBV frameshift signal and is fully functional in frameshifting (Brierley et al ., 1992) . It differs from the wild-type in a number of ways; a termination codon (UGA) is present immediately downstream of the slippery sequence (UUUAAAC, boxed) to terminate zero frame ribosomes, loop 2 of the pseudoknot contains 8 rather than 32 nt, the G·A mismatched pair in stem 1 of the wild-type pseudoknot is replaced by a U·G pair, the G nucleotide of loop 1 is replaced by a C nucleotide and, finally, the minimal pseudoknot has no stop codons. Plasmid pKA1 was derived from pFScass 6 by deletion mutagenesis (see Materials and Methods). The type and number of bases in the loops and stem 2 remained unaltered. The predicted stability of stem 1 of each construct is shown (calculated according to the rules by Turner et al ., 1988 , using a loop length of 8 nt; see the text).
    Bacteriophage Sp6 Rna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs small rna library preparation kit
    Strand-specific small <t>RNA</t> coverage ( y -axis; log2) of the genes ND169 (A and B), DCR1 (C and D) in the respective serotype-knockdown library is shown. (E) A bar plot showing the normalized sRNA read counts ( y -axis) of the knocked down RNAi genes in the respective serotype-knockdown samples ( x -axis; library). Just for this figure element, normalization was carried out using only total count scaling (TCS), i.e. knockdown-associated regions were not removed. (F) Fold change of gene expression (TPM in knockdown vs. TPM of wild-type of mRNAs targeted by primary siRNAs in both serotypes).
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    The Monarch RNA Cleanup Columns 500 µg are a component of the Monarch RNA Cleanup Kit 500 µg NEB T2050 and can be used to purify up to 500 µg
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    RNA isolation and RT-qPCR in microgravity. (A) Photo of SPM. (B) Typical RNA quality from SPM with E . coli (left panel) and mouse liver (right panel), control Qiagen (left lane) SPM (right lane). Center panel shows RNA quality from the 1 g control (left lane) and the returned microgravity sample from ISS (right lane). (C-E) Scatter plots with jitter of the microgravity and 1 g control E . coli singleplex (C), duplex (D) and triplex (E) reactions. One outlier is indicated by the open marker in C. One of the microgravity triplex tubes did not give a dnaK-FAM signal (E). (F-H) Scatter plots with jitter of the microgravity and 1 g control mouse liver singleplex (F), duplex (G) and triplex (H) reactions. One outlier from the microgravity triplex fn1 plot is indicated by the open marker and no gapdh-FAM signal was seen in the microgravity triplex reactions (H).

    Journal: PLoS ONE

    Article Title: Microgravity validation of a novel system for RNA isolation and multiplex quantitative real time PCR analysis of gene expression on the International Space Station

    doi: 10.1371/journal.pone.0183480

    Figure Lengend Snippet: RNA isolation and RT-qPCR in microgravity. (A) Photo of SPM. (B) Typical RNA quality from SPM with E . coli (left panel) and mouse liver (right panel), control Qiagen (left lane) SPM (right lane). Center panel shows RNA quality from the 1 g control (left lane) and the returned microgravity sample from ISS (right lane). (C-E) Scatter plots with jitter of the microgravity and 1 g control E . coli singleplex (C), duplex (D) and triplex (E) reactions. One outlier is indicated by the open marker in C. One of the microgravity triplex tubes did not give a dnaK-FAM signal (E). (F-H) Scatter plots with jitter of the microgravity and 1 g control mouse liver singleplex (F), duplex (G) and triplex (H) reactions. One outlier from the microgravity triplex fn1 plot is indicated by the open marker and no gapdh-FAM signal was seen in the microgravity triplex reactions (H).

    Article Snippet: Genomic and gene expression assays for E . coli (dnaK , rpoA and srIR ) and mouse tissue (gapdh, rpl19, and fn1) were validated using genomic E . coli DNA and RNA from E . coli K12 TB1 (New England Biolabs Inc, Ipswich, MA) and whole livers from male C57BL/6 12–16 week perfused/snap frozen (Charles River, Wilmington, MA).

    Techniques: Isolation, Quantitative RT-PCR, Marker

    Usp9x -mutant embryos arrest at E9.5–E11.5 and display defective repression of early lineage programs marked by H3K27me3. a Genetic cross to delete Usp9x in epiblast derivatives of postimplantation embryos. Quantification of recovered (live) male embryos at several postimplantation stages (right). b Sample images and quantification of control and mutant embryo phenotypes. Relative to controls (left), E9.5 embryos show variable developmental delay, with closed arrow indicating an open anterior neuropore. E11.5 embryos show a range of phenotypes, from hemorrhage to severe delay and death (tally includes dead embryos). Open arrow indicates pericardial edema. Scale bars = 250 µm (E9.5), 2.8 mm (E11.5), with N indicated. c MA plots of expression changes by RNA-seq in two litters of Usp9x mutants versus controls (at E8.5). 3 mutants and 3 controls were sequenced per litter ( N = 12 embryos total; see Supplementary Fig. 3b-d ). d Enrichr analysis of the top-enriched transcription factors (TF) that bind to the genes upregulated in Usp9x -mutant embryos in various cell types. e Expression of the 71 genes upregulated in Usp9x mutants during wild-type development 56 . FC , Fold-change relative to E6.5 embryos. f Distribution and boxplot quantification of H3K27me3 levels 59 over the promoters of genes upregulated in Usp9x mutants (10 kb upstream, 1 kb downstream of TSS). g Representative genome browser tracks of H3K27me3 in wild-type embryos (E6.5–E8.5) at the Nodal locus 59 . Known enhancer elements are highlighted and show gains of H3K27me3. h Nodal mRNA expression in wild-type development 56 . i Nodal mRNA expression in E8.5 Usp9x- mutant or control embryos. Boxplot hinges show the first and third quartiles, whiskers show ±1.5*IQR and center line shows median of 2–3 biological replicates ( e , f ). Data are representative of 2–3 biological replicates ( g ), mean ± s.e.m. of 2–3 biological replicates ( h ), or 6 biological replicates ( i ). P -values by χ 2 test ( a , b ), Fisher’s exact test ( d ), two-tailed Student’s t -tests with Welch’s correction ( e , i ), two-tailed Wilcoxon rank-sum tests ( f ), and ANOVA with Dunnett’s multiple comparison test to E6.5 ( h ). χ 2 = 19.78 ( a ), 85.19 ( b , top), 147.8 ( b , bottom).

    Journal: Nature Communications

    Article Title: The deubiquitinase Usp9x regulates PRC2-mediated chromatin reprogramming during mouse development

    doi: 10.1038/s41467-021-21910-0

    Figure Lengend Snippet: Usp9x -mutant embryos arrest at E9.5–E11.5 and display defective repression of early lineage programs marked by H3K27me3. a Genetic cross to delete Usp9x in epiblast derivatives of postimplantation embryos. Quantification of recovered (live) male embryos at several postimplantation stages (right). b Sample images and quantification of control and mutant embryo phenotypes. Relative to controls (left), E9.5 embryos show variable developmental delay, with closed arrow indicating an open anterior neuropore. E11.5 embryos show a range of phenotypes, from hemorrhage to severe delay and death (tally includes dead embryos). Open arrow indicates pericardial edema. Scale bars = 250 µm (E9.5), 2.8 mm (E11.5), with N indicated. c MA plots of expression changes by RNA-seq in two litters of Usp9x mutants versus controls (at E8.5). 3 mutants and 3 controls were sequenced per litter ( N = 12 embryos total; see Supplementary Fig. 3b-d ). d Enrichr analysis of the top-enriched transcription factors (TF) that bind to the genes upregulated in Usp9x -mutant embryos in various cell types. e Expression of the 71 genes upregulated in Usp9x mutants during wild-type development 56 . FC , Fold-change relative to E6.5 embryos. f Distribution and boxplot quantification of H3K27me3 levels 59 over the promoters of genes upregulated in Usp9x mutants (10 kb upstream, 1 kb downstream of TSS). g Representative genome browser tracks of H3K27me3 in wild-type embryos (E6.5–E8.5) at the Nodal locus 59 . Known enhancer elements are highlighted and show gains of H3K27me3. h Nodal mRNA expression in wild-type development 56 . i Nodal mRNA expression in E8.5 Usp9x- mutant or control embryos. Boxplot hinges show the first and third quartiles, whiskers show ±1.5*IQR and center line shows median of 2–3 biological replicates ( e , f ). Data are representative of 2–3 biological replicates ( g ), mean ± s.e.m. of 2–3 biological replicates ( h ), or 6 biological replicates ( i ). P -values by χ 2 test ( a , b ), Fisher’s exact test ( d ), two-tailed Student’s t -tests with Welch’s correction ( e , i ), two-tailed Wilcoxon rank-sum tests ( f ), and ANOVA with Dunnett’s multiple comparison test to E6.5 ( h ). χ 2 = 19.78 ( a ), 85.19 ( b , top), 147.8 ( b , bottom).

    Article Snippet: One microgram of total RNA was used for mRNA isolation and library preparation using the NEBNext Ultra II Directional Library Prep Kit for Illumina with the mRNA Magnetic Isolation Module, per manufacturer’s instructions (New England Biolabs, NEB #E7420S and #E7490S).

    Techniques: Mutagenesis, Expressing, RNA Sequencing Assay, Two Tailed Test

    Usp9x promotes ES cell self-renewal and a transcriptional signature of preimplantation linked to PRC2 activity. a Schematic of an auxin-inducible degron (AID) system for acute Usp9x depletion in mouse embryonic stem (ES) cells with representative flow cytometry plot of GFP (AID-Usp9x) expression in Usp9x-low and Usp9x-high ES cells. Right: western blot of endogenous Usp9x level in sorted cell fractions (see Supplementary Fig. 1b ). b Quantification and representative images of colony formation assays. Usp9x-low ES cells display a self-renewal deficit. AP , Alkaline Phosphatase. c Principal Component (PC) Analysis of gene expression by RNA-seq. 8 h : 8 h auxin. No auxin : AID-Usp9x cells with vehicle treatment. 48 h : 8 h auxin followed by 48 h recovery without auxin. Flag : Flag-Usp9x cells after 8 h auxin and 48 h recovery. d The transcriptional signatures of Usp9x-high or Usp9x-low ES cells correlate with different stages of peri-implantation development by Gene Set Enrichment Analysis (GSEA). Genes differentially expressed between Usp9x-high or Usp9x-low ES cells and controls were used in each case. See Methods for references. DE, differentially expressed (relative to controls); NS, not significant (FDR > 0.05); NES, Normalized Enrichment Score. e Usp9x mRNA expression in the epiblast declines from pre- to postimplantation 36 , 37 . f Flow cytometry plot measuring median fluorescence intensity of GFP (Usp9x expression) in Usp9x-high and Usp9x-low ES cells after 8 h auxin treatment and 48 h recovery (without auxin). g Fold-change in expression of all genes at 48 h relative to control cells, showing hypotranscription in Usp9x-high ES cells and hypertranscription in Usp9x-low ES cells. h Heatmaps with summary profile plot of Suz12 binding (data from wild-type ES cells 43 ) over the genes upregulated in Usp9x-low cells or a random subset ( N = 1310). i Boxplots showing repression (in Usp9x-high) or induction (in Usp9x-low) of Suz12 target genes 57 , compared to a random subset ( N = 3350). Western blots represent at least two biological replicates ( a ). Data are mean ± s.d. of four replicates from two independent experiments ( b ), mean ± s.d. of 3–4 replicates ( e ), representative of three experiments ( f , h ). Boxplot hinges ( g , i ) show the first and third quartiles, whiskers show ±1.5*inter-quartile range (IQR) and center line shows median of three biological replicates. **** P

    Journal: Nature Communications

    Article Title: The deubiquitinase Usp9x regulates PRC2-mediated chromatin reprogramming during mouse development

    doi: 10.1038/s41467-021-21910-0

    Figure Lengend Snippet: Usp9x promotes ES cell self-renewal and a transcriptional signature of preimplantation linked to PRC2 activity. a Schematic of an auxin-inducible degron (AID) system for acute Usp9x depletion in mouse embryonic stem (ES) cells with representative flow cytometry plot of GFP (AID-Usp9x) expression in Usp9x-low and Usp9x-high ES cells. Right: western blot of endogenous Usp9x level in sorted cell fractions (see Supplementary Fig. 1b ). b Quantification and representative images of colony formation assays. Usp9x-low ES cells display a self-renewal deficit. AP , Alkaline Phosphatase. c Principal Component (PC) Analysis of gene expression by RNA-seq. 8 h : 8 h auxin. No auxin : AID-Usp9x cells with vehicle treatment. 48 h : 8 h auxin followed by 48 h recovery without auxin. Flag : Flag-Usp9x cells after 8 h auxin and 48 h recovery. d The transcriptional signatures of Usp9x-high or Usp9x-low ES cells correlate with different stages of peri-implantation development by Gene Set Enrichment Analysis (GSEA). Genes differentially expressed between Usp9x-high or Usp9x-low ES cells and controls were used in each case. See Methods for references. DE, differentially expressed (relative to controls); NS, not significant (FDR > 0.05); NES, Normalized Enrichment Score. e Usp9x mRNA expression in the epiblast declines from pre- to postimplantation 36 , 37 . f Flow cytometry plot measuring median fluorescence intensity of GFP (Usp9x expression) in Usp9x-high and Usp9x-low ES cells after 8 h auxin treatment and 48 h recovery (without auxin). g Fold-change in expression of all genes at 48 h relative to control cells, showing hypotranscription in Usp9x-high ES cells and hypertranscription in Usp9x-low ES cells. h Heatmaps with summary profile plot of Suz12 binding (data from wild-type ES cells 43 ) over the genes upregulated in Usp9x-low cells or a random subset ( N = 1310). i Boxplots showing repression (in Usp9x-high) or induction (in Usp9x-low) of Suz12 target genes 57 , compared to a random subset ( N = 3350). Western blots represent at least two biological replicates ( a ). Data are mean ± s.d. of four replicates from two independent experiments ( b ), mean ± s.d. of 3–4 replicates ( e ), representative of three experiments ( f , h ). Boxplot hinges ( g , i ) show the first and third quartiles, whiskers show ±1.5*inter-quartile range (IQR) and center line shows median of three biological replicates. **** P

    Article Snippet: One microgram of total RNA was used for mRNA isolation and library preparation using the NEBNext Ultra II Directional Library Prep Kit for Illumina with the mRNA Magnetic Isolation Module, per manufacturer’s instructions (New England Biolabs, NEB #E7420S and #E7490S).

    Techniques: Activity Assay, Flow Cytometry, Expressing, Western Blot, RNA Sequencing Assay, Fluorescence, Binding Assay

    The frameshift reporter construct pKA1. (a) Plasmid pKA1 was derived from pFScass 6 (Brierley et al ., 1992) by site-directed mutagenesis (see Materials and Methods). pKA1 contains a truncated version of the minimal IBV pseudoknot (white box) cloned into a reporter gene, the influenza PB2 gene (shaded boxes). Linearisation of the plasmid with Bam H1 and in vitro transcription using SP6 RNA polymerase yields an mRNA (2.4kb) that, when translated in RRL, is predicted to produce a 19 kDa non-frameshift product corresponding to ribosomes that terminate at the UGA termination codon (located immediately downstream of the slippery sequence UUUAAAC, shaded), and a 22 kDa −1 frameshift product. The 0-frame and −2/+1 frames are also open (to some extent) in this construct. Ribosomes which enter these frames produce 28 kDa and 85 kDa products respectively. A bacteriophage T7 promoter is present just upstream of the frameshift region; this promoter is employed to generate short, pseudoknot-containing transcripts from Bst NI-digested templates for secondary structure analysis. (b) The wild-type (wt) IBV, the minimal IBV and the pKA1 pseudoknots (PK). The minimal IBV frameshift signal present in pFScass 6 is based on the wild-type IBV frameshift signal and is fully functional in frameshifting (Brierley et al ., 1992) . It differs from the wild-type in a number of ways; a termination codon (UGA) is present immediately downstream of the slippery sequence (UUUAAAC, boxed) to terminate zero frame ribosomes, loop 2 of the pseudoknot contains 8 rather than 32 nt, the G·A mismatched pair in stem 1 of the wild-type pseudoknot is replaced by a U·G pair, the G nucleotide of loop 1 is replaced by a C nucleotide and, finally, the minimal pseudoknot has no stop codons. Plasmid pKA1 was derived from pFScass 6 by deletion mutagenesis (see Materials and Methods). The type and number of bases in the loops and stem 2 remained unaltered. The predicted stability of stem 1 of each construct is shown (calculated according to the rules by Turner et al ., 1988 , using a loop length of 8 nt; see the text).

    Journal: Journal of Molecular Biology

    Article Title: The role of RNA pseudoknot stem 1 length in the promotion of efficient −1 ribosomal frameshifting 1

    doi: 10.1006/jmbi.1999.2688

    Figure Lengend Snippet: The frameshift reporter construct pKA1. (a) Plasmid pKA1 was derived from pFScass 6 (Brierley et al ., 1992) by site-directed mutagenesis (see Materials and Methods). pKA1 contains a truncated version of the minimal IBV pseudoknot (white box) cloned into a reporter gene, the influenza PB2 gene (shaded boxes). Linearisation of the plasmid with Bam H1 and in vitro transcription using SP6 RNA polymerase yields an mRNA (2.4kb) that, when translated in RRL, is predicted to produce a 19 kDa non-frameshift product corresponding to ribosomes that terminate at the UGA termination codon (located immediately downstream of the slippery sequence UUUAAAC, shaded), and a 22 kDa −1 frameshift product. The 0-frame and −2/+1 frames are also open (to some extent) in this construct. Ribosomes which enter these frames produce 28 kDa and 85 kDa products respectively. A bacteriophage T7 promoter is present just upstream of the frameshift region; this promoter is employed to generate short, pseudoknot-containing transcripts from Bst NI-digested templates for secondary structure analysis. (b) The wild-type (wt) IBV, the minimal IBV and the pKA1 pseudoknots (PK). The minimal IBV frameshift signal present in pFScass 6 is based on the wild-type IBV frameshift signal and is fully functional in frameshifting (Brierley et al ., 1992) . It differs from the wild-type in a number of ways; a termination codon (UGA) is present immediately downstream of the slippery sequence (UUUAAAC, boxed) to terminate zero frame ribosomes, loop 2 of the pseudoknot contains 8 rather than 32 nt, the G·A mismatched pair in stem 1 of the wild-type pseudoknot is replaced by a U·G pair, the G nucleotide of loop 1 is replaced by a C nucleotide and, finally, the minimal pseudoknot has no stop codons. Plasmid pKA1 was derived from pFScass 6 by deletion mutagenesis (see Materials and Methods). The type and number of bases in the loops and stem 2 remained unaltered. The predicted stability of stem 1 of each construct is shown (calculated according to the rules by Turner et al ., 1988 , using a loop length of 8 nt; see the text).

    Article Snippet: In vitro transcription reactions employing the bacteriophage SP6 RNA polymerase were carried out essentially as described by and included the synthetic cap structure 7meGpppG (New England Biolabs) to generate capped mRNA.

    Techniques: Construct, Plasmid Preparation, Derivative Assay, Mutagenesis, Clone Assay, In Vitro, Sequencing, Functional Assay

    Strand-specific small RNA coverage ( y -axis; log2) of the genes ND169 (A and B), DCR1 (C and D) in the respective serotype-knockdown library is shown. (E) A bar plot showing the normalized sRNA read counts ( y -axis) of the knocked down RNAi genes in the respective serotype-knockdown samples ( x -axis; library). Just for this figure element, normalization was carried out using only total count scaling (TCS), i.e. knockdown-associated regions were not removed. (F) Fold change of gene expression (TPM in knockdown vs. TPM of wild-type of mRNAs targeted by primary siRNAs in both serotypes).

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    Article Title: Feeding exogenous dsRNA interferes with endogenous sRNA accumulation in Paramecium

    doi: 10.1093/dnares/dsaa005

    Figure Lengend Snippet: Strand-specific small RNA coverage ( y -axis; log2) of the genes ND169 (A and B), DCR1 (C and D) in the respective serotype-knockdown library is shown. (E) A bar plot showing the normalized sRNA read counts ( y -axis) of the knocked down RNAi genes in the respective serotype-knockdown samples ( x -axis; library). Just for this figure element, normalization was carried out using only total count scaling (TCS), i.e. knockdown-associated regions were not removed. (F) Fold change of gene expression (TPM in knockdown vs. TPM of wild-type of mRNAs targeted by primary siRNAs in both serotypes).

    Article Snippet: After re-isolation of the sRNAs by extraction in 0.3 M NaCl, sRNAs were precipitated and we used the NEB Small RNA library preparation Kit (New England Biolabs) with elongated 3´-adapter ligation to limit biases against 2´-O-methylated siRNAs.

    Techniques: Expressing