monarch rna cleanup kit  (New England Biolabs)


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    Monarch RNA Cleanup Columns 10 µg
    Description:
    The Monarch RNA Cleanup Columns 10 µg are a component of the Monarch RNA Cleanup Kit 10 µg NEB T2030 and can be used to purify and concentrate up to 10 µg of RNA from enzymatic reactions including DNase treatment NEB M0303 labeling and capping The columns are designed without the use of a retaining ring ensuring no buffer retention and no carryover of contaminants RNA can be eluted in as little as 6 μl for and is ready for use in a variety of downstream applications including RT PCR and RNA Library Prep for NGS
    Catalog Number:
    T2037L
    Price:
    200
    Category:
    RNA Purification Kit Components
    Size:
    100 columns
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    Structured Review

    New England Biolabs monarch rna cleanup kit
    Monarch RNA Cleanup Columns 10 µg
    The Monarch RNA Cleanup Columns 10 µg are a component of the Monarch RNA Cleanup Kit 10 µg NEB T2030 and can be used to purify and concentrate up to 10 µg of RNA from enzymatic reactions including DNase treatment NEB M0303 labeling and capping The columns are designed without the use of a retaining ring ensuring no buffer retention and no carryover of contaminants RNA can be eluted in as little as 6 μl for and is ready for use in a variety of downstream applications including RT PCR and RNA Library Prep for NGS
    https://www.bioz.com/result/monarch rna cleanup kit/product/New England Biolabs
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    monarch rna cleanup kit - by Bioz Stars, 2021-04
    98/100 stars

    Images

    1) Product Images from "In vitro transcription using psychrophilic phage VSW-3 RNA polymerase"

    Article Title: In vitro transcription using psychrophilic phage VSW-3 RNA polymerase

    Journal: bioRxiv

    doi: 10.1101/2020.09.14.297226

    VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized pUC19 plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The RNA yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.
    Figure Legend Snippet: VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized pUC19 plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The RNA yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.

    Techniques Used: SDS Page, Purification, Staining, Plasmid Preparation, Sequencing

    2) Product Images from "Synthesis of low immunogenicity RNA with high-temperature in vitro transcription"

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    Journal: bioRxiv

    doi: 10.1101/815092

    Template-encoded Poly(A) tailing reduces antisense by-product formation. A) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30 bp, 60 bp, 120 bp) of poly-T sequence at 3’-end under standard conditions (5 mM rNTPs, 37°C for 1 hour). B) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly-T (60 bp and 120 bp) sequence at the 3’-end. IVT reactions were performed at 37°C or 50°C.
    Figure Legend Snippet: Template-encoded Poly(A) tailing reduces antisense by-product formation. A) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30 bp, 60 bp, 120 bp) of poly-T sequence at 3’-end under standard conditions (5 mM rNTPs, 37°C for 1 hour). B) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly-T (60 bp and 120 bp) sequence at the 3’-end. IVT reactions were performed at 37°C or 50°C.

    Techniques Used: Nucleic Acid Electrophoresis, Synthesized, Sequencing

    Truncation of the 3’-end of the 512B DNA templates results in reduction of the antisense RNA by-product formation. Immunoblot (with J2 antibody; 1:5000; Scicons) and native gel electrophoresis analyses of in vitro transcription reactions performed on 512B template with 3’-end truncations (50 and 200 base pairs). In vitro transcription reactions were performed with TsT7-1 at 37°C or 50°C.
    Figure Legend Snippet: Truncation of the 3’-end of the 512B DNA templates results in reduction of the antisense RNA by-product formation. Immunoblot (with J2 antibody; 1:5000; Scicons) and native gel electrophoresis analyses of in vitro transcription reactions performed on 512B template with 3’-end truncations (50 and 200 base pairs). In vitro transcription reactions were performed with TsT7-1 at 37°C or 50°C.

    Techniques Used: Nucleic Acid Electrophoresis, In Vitro

    3) Product Images from "In vitro transcription using psychrophilic phage VSW-3 RNA polymerase"

    Article Title: In vitro transcription using psychrophilic phage VSW-3 RNA polymerase

    Journal: bioRxiv

    doi: 10.1101/2020.09.14.297226

    Optimal VSW-3 RNAP IVT conditions. (A) Screening for the optimal Mg 2+ /NTP concentration in the presence of 1 mM DTT. RNA yield with various optimal Mg 2+ /NTP concentration combination was further compared (gel in the dotted box). The stability of the optimal VSW-3 RNAP IVT buffer with 1 mM DTT was examined (gel in the solid box). (B) Screening for the optimal DTT/ Mg 2+ concentration for the stable and high-yield VSW-3 RNAP IVT buffer. The stability of the high-yield VSW-3 RNAP IVT buffer containing 16 mM Mg 2+ , 4 mM NTP and 5 mM DTT was examined (gel in the solid box). (C) The optimal reaction temperature of VSW-3 RNAP (25°C) for maximum run-off RNA yield. (D) The optimal enzyme concentration of VSW-3 RNAP (0.15 μM) for maximum run-off RNA yield. (E) Optimal IVT yield of VSW-3 RNAP with various reaction temperature/incubation time combinations. The maximum run-off RNA yield was obtained at 25°C for 12 hours. (F) Gray-scale quantitation of the run-off RNA transcripts in gel (E) . Diagram was made using GraphPad Prism. In all gels the bands corresponding to DNA templates were indicated by empty stars and the bands corresponding to run-off RNA transcripts were indicated by filled stars.
    Figure Legend Snippet: Optimal VSW-3 RNAP IVT conditions. (A) Screening for the optimal Mg 2+ /NTP concentration in the presence of 1 mM DTT. RNA yield with various optimal Mg 2+ /NTP concentration combination was further compared (gel in the dotted box). The stability of the optimal VSW-3 RNAP IVT buffer with 1 mM DTT was examined (gel in the solid box). (B) Screening for the optimal DTT/ Mg 2+ concentration for the stable and high-yield VSW-3 RNAP IVT buffer. The stability of the high-yield VSW-3 RNAP IVT buffer containing 16 mM Mg 2+ , 4 mM NTP and 5 mM DTT was examined (gel in the solid box). (C) The optimal reaction temperature of VSW-3 RNAP (25°C) for maximum run-off RNA yield. (D) The optimal enzyme concentration of VSW-3 RNAP (0.15 μM) for maximum run-off RNA yield. (E) Optimal IVT yield of VSW-3 RNAP with various reaction temperature/incubation time combinations. The maximum run-off RNA yield was obtained at 25°C for 12 hours. (F) Gray-scale quantitation of the run-off RNA transcripts in gel (E) . Diagram was made using GraphPad Prism. In all gels the bands corresponding to DNA templates were indicated by empty stars and the bands corresponding to run-off RNA transcripts were indicated by filled stars.

    Techniques Used: Concentration Assay, Incubation, Quantitation Assay

    Response of ssRNAPs to Class II terminator. (A) Using PCR-amplified templates for cas9-RNA IVT, obvious abortive RNA transcripts were observed for T7 RNAP and Syn5 RNAP but not VSW-3 RNAP (top gel). 3’-RACE revealed that the T7 RNAP transcription was terminated 9 nt downstream of a Class II terminator “ATCTGTT” (bottom sequencing result). (B) VSW-3 RNAP IVT was not terminated (no additional bands comparing lane 2 with lane 1) when a Class II terminator “ATCTGTT” was inserted into the middle of the copGFP RNA coding sequence.
    Figure Legend Snippet: Response of ssRNAPs to Class II terminator. (A) Using PCR-amplified templates for cas9-RNA IVT, obvious abortive RNA transcripts were observed for T7 RNAP and Syn5 RNAP but not VSW-3 RNAP (top gel). 3’-RACE revealed that the T7 RNAP transcription was terminated 9 nt downstream of a Class II terminator “ATCTGTT” (bottom sequencing result). (B) VSW-3 RNAP IVT was not terminated (no additional bands comparing lane 2 with lane 1) when a Class II terminator “ATCTGTT” was inserted into the middle of the copGFP RNA coding sequence.

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing

    RNA 3’ extension and RdRp activity of T7 and VSW-3 RNAP. (A) The secondary structure of a sgRNA predicted with RNAfold software. (B) IVT synthesis of a sgRNA (targeting eGFP) by VSW-3 and T7 RNAP. (C) 3’-RACE of the sgRNAs transcripts from T7 and VSW-3 RNAP IVT. Only the 3’ region (red sequence on the top) of the full sgRNA in sequencing results was shown. The length of each sequence was noted. The sequences matching the exact run-off sgRNA (103 nt) was indicated by red stars. (D) Schematic showing the mechanism and origin (3’ self-templated extension by the RdRp activity of T7 RNAP) of the 16 nt 3’-extension in T7 RNAP products as in (C) . (E) T7 but not VSW-3 RNAP retains the RdRp activity to extend purified sgRNA (with terminal primer/template structure).
    Figure Legend Snippet: RNA 3’ extension and RdRp activity of T7 and VSW-3 RNAP. (A) The secondary structure of a sgRNA predicted with RNAfold software. (B) IVT synthesis of a sgRNA (targeting eGFP) by VSW-3 and T7 RNAP. (C) 3’-RACE of the sgRNAs transcripts from T7 and VSW-3 RNAP IVT. Only the 3’ region (red sequence on the top) of the full sgRNA in sequencing results was shown. The length of each sequence was noted. The sequences matching the exact run-off sgRNA (103 nt) was indicated by red stars. (D) Schematic showing the mechanism and origin (3’ self-templated extension by the RdRp activity of T7 RNAP) of the 16 nt 3’-extension in T7 RNAP products as in (C) . (E) T7 but not VSW-3 RNAP retains the RdRp activity to extend purified sgRNA (with terminal primer/template structure).

    Techniques Used: Activity Assay, Software, Sequencing, Purification

    VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized pUC19 plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The RNA yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.
    Figure Legend Snippet: VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized pUC19 plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The RNA yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.

    Techniques Used: SDS Page, Purification, Staining, Plasmid Preparation, Sequencing

    dsRNA by-products from T7 and VSW-3 RNAP IVT. (A) DNA templates for the IVT synthesis of various RNA as indicated on top of the gel. For each RNA, there are two DNA templates differ only in the promoter region to serve for VSW-3 and T7 RNAP IVT, respectively. DNA concentration and purity were compared in 1.5% agarose gel stained with ethidium bromide. (B) After template DNA was removed by DNase I treatment and purified with Monarch RNA Cleanup kit, 1μg of sox7, tdTomato, copGFP and Cas9 RNA transcribed by VSW-3 RNAP and T7 RNAP were analyzed in 1.5% agarose gel stained with ethidium bromide. The white and black colors for bands and background were converted in this gel picture to make the weak double-stranded and abortive RNA bands clearer. (C) Dot blot analysis of the RNA products (each 200 ng) as in (B) by VSW-3 RNAP and T7 RNAP with J2 monoclonal antibody. A prepared dsRNA (351 bp) was applied as quantitative standard (0.1 ng, 0.25 ng, 0.5 ng, 1.0 ng). (D) The gray value measurement and calculation of the X film image (top image in (C) ) by Image J software demonstrating the level of dsRNA contamination in T7 and VSW-3 RNAP transcripts.
    Figure Legend Snippet: dsRNA by-products from T7 and VSW-3 RNAP IVT. (A) DNA templates for the IVT synthesis of various RNA as indicated on top of the gel. For each RNA, there are two DNA templates differ only in the promoter region to serve for VSW-3 and T7 RNAP IVT, respectively. DNA concentration and purity were compared in 1.5% agarose gel stained with ethidium bromide. (B) After template DNA was removed by DNase I treatment and purified with Monarch RNA Cleanup kit, 1μg of sox7, tdTomato, copGFP and Cas9 RNA transcribed by VSW-3 RNAP and T7 RNAP were analyzed in 1.5% agarose gel stained with ethidium bromide. The white and black colors for bands and background were converted in this gel picture to make the weak double-stranded and abortive RNA bands clearer. (C) Dot blot analysis of the RNA products (each 200 ng) as in (B) by VSW-3 RNAP and T7 RNAP with J2 monoclonal antibody. A prepared dsRNA (351 bp) was applied as quantitative standard (0.1 ng, 0.25 ng, 0.5 ng, 1.0 ng). (D) The gray value measurement and calculation of the X film image (top image in (C) ) by Image J software demonstrating the level of dsRNA contamination in T7 and VSW-3 RNAP transcripts.

    Techniques Used: Concentration Assay, Agarose Gel Electrophoresis, Staining, Purification, Dot Blot, Software

    4) Product Images from "An RNA polymerase ribozyme that synthesizes its own ancestor"

    Article Title: An RNA polymerase ribozyme that synthesizes its own ancestor

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

    doi: 10.1073/pnas.1914282117

    In vitro evolution of the 38-6 RNA polymerase ribozyme. ( A ) Scheme for selective amplification of polymerase ribozymes that synthesize a functional hammerhead ribozyme. (1) Attachment to the polymerase of an RNA primer (magenta), biotin (green), and the RNA substrate (orange) to be cleaved by the hammerhead. (2) Hybridization of the primer to an RNA template (brown) that encodes the hammerhead. (3) Extension of the primer by polymerization of NTPs (cyan), followed by biotin capture on streptavidin magnetic beads (gray). (4) Cleavage of the attached RNA substrate by the hammerhead, releasing the polymerase from the beads. (5) Recovery of functional polymerases. (6) Reverse transcription and PCR amplification. (7) Transcription to generate progeny polymerases. ( B ) Sequence and secondary structure of the hammerhead ribozyme (cyan), together with the primer used to initiate its synthesis and the RNA substrate. The arrow indicates the site of cleavage. ( C ). Stem elements P3–P7 within the core domain are labeled.
    Figure Legend Snippet: In vitro evolution of the 38-6 RNA polymerase ribozyme. ( A ) Scheme for selective amplification of polymerase ribozymes that synthesize a functional hammerhead ribozyme. (1) Attachment to the polymerase of an RNA primer (magenta), biotin (green), and the RNA substrate (orange) to be cleaved by the hammerhead. (2) Hybridization of the primer to an RNA template (brown) that encodes the hammerhead. (3) Extension of the primer by polymerization of NTPs (cyan), followed by biotin capture on streptavidin magnetic beads (gray). (4) Cleavage of the attached RNA substrate by the hammerhead, releasing the polymerase from the beads. (5) Recovery of functional polymerases. (6) Reverse transcription and PCR amplification. (7) Transcription to generate progeny polymerases. ( B ) Sequence and secondary structure of the hammerhead ribozyme (cyan), together with the primer used to initiate its synthesis and the RNA substrate. The arrow indicates the site of cleavage. ( C ). Stem elements P3–P7 within the core domain are labeled.

    Techniques Used: In Vitro, Amplification, Functional Assay, Hybridization, Magnetic Beads, Polymerase Chain Reaction, Sequencing, Labeling

    5) Product Images from "Synthesis of low immunogenicity RNA with high-temperature in vitro transcription"

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    Journal: RNA

    doi: 10.1261/rna.073858.119

    Template-encoded poly(A) tailing reduces antisense by-product formation. ( A ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30, 60, 120 bp) of poly(T) sequence at 3′ end under standard conditions. ( B ) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly(T) (60 and 120 bp) sequence at the 3′ end. IVT reactions were performed at 37°C or 50°C.
    Figure Legend Snippet: Template-encoded poly(A) tailing reduces antisense by-product formation. ( A ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30, 60, 120 bp) of poly(T) sequence at 3′ end under standard conditions. ( B ) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly(T) (60 and 120 bp) sequence at the 3′ end. IVT reactions were performed at 37°C or 50°C.

    Techniques Used: Nucleic Acid Electrophoresis, Synthesized, Sequencing

    6) Product Images from "Klebsiella Phage KP34 RNA Polymerase and Its Use in RNA Synthesis"

    Article Title: Klebsiella Phage KP34 RNA Polymerase and Its Use in RNA Synthesis

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2019.02487

    Identification of the KP34 RNAP promoter. (A) The DNA fragment (Template 1) containing previously predicated promoters failed to serve as transcription template for KP34 RNAP to produce RNA in vitro , while the DNA fragment covering the RNAP gene and downstream gap region (Template 2) was active as transcription template. RNA products are shown as bright bands on the 2% TAE agarose gel. (B) 5′-RACE analysis of the position of transcription initiation. 5′ sequence of KP34 transcripts were matched to KP34 genome. Major sequences were in solid box and minor sequences in dotted box. Their upstream region containing putative promoters is shown in bold. (C) Comparison of run-off RNA synthesis by T7 and KP34 RNAP under the control of various promoters. A DNA template containing a T7 promoter (5′-TAATACGACTCACTATA-3′) was incubated with 100 nM T7 RNAP, and three DNA templates containing either a KP34 strong promoter S1 (5′-TAATGTTACAGGAGTA-3′), a KP34 strong promoter S2 (5′-TGATGTTACAGGAGTA-3′), or a KP34 weak promoter W (5′-ACTTTGGACATCCG TCAAGT-3′) were incubated with 100 nM KP34 RNAP to direct to the transcription of their downstream sequence that encodes the same 37 nt RNA. [α-32P]ATP was added into reactions for imaging and visualization. Reaction products were separated by a 25% TBE-Urea denaturing gel. (D) Identification of the full KP34 strong promoter. A KP34 strong promoter S1 (5′-TAATGTTACAGGAGTA-3′) or the 3′ 14 nt common sequence of the two KP34 strong promoters (5′-ATGTTA CAGGAGTA-3′) was inserted into plasmid pUC19 to direct the transcription of their downstream sequences, respectively. A run-off transcript of ∼2700 nt and a terminated transcript of ∼1000 nt (terminated by a predictable T7 class I hairpin terminator structure) were expected from the linearized form of these plasmids if the inserted promoter is sufficient to direct transcription by KP34 RNAP. M: ssRNA Ladder. (E) KP34 promoters in the genome (location of strong promoter (S) pointed by solid arrow and weak promoter (W) by dotted arrow) and comparison of typical ssRNAP promoters. Conserved sequence among ssRNAP promoters are in bold and those homologous between Syn5 promoter and KP34 weak promoter are underlined.
    Figure Legend Snippet: Identification of the KP34 RNAP promoter. (A) The DNA fragment (Template 1) containing previously predicated promoters failed to serve as transcription template for KP34 RNAP to produce RNA in vitro , while the DNA fragment covering the RNAP gene and downstream gap region (Template 2) was active as transcription template. RNA products are shown as bright bands on the 2% TAE agarose gel. (B) 5′-RACE analysis of the position of transcription initiation. 5′ sequence of KP34 transcripts were matched to KP34 genome. Major sequences were in solid box and minor sequences in dotted box. Their upstream region containing putative promoters is shown in bold. (C) Comparison of run-off RNA synthesis by T7 and KP34 RNAP under the control of various promoters. A DNA template containing a T7 promoter (5′-TAATACGACTCACTATA-3′) was incubated with 100 nM T7 RNAP, and three DNA templates containing either a KP34 strong promoter S1 (5′-TAATGTTACAGGAGTA-3′), a KP34 strong promoter S2 (5′-TGATGTTACAGGAGTA-3′), or a KP34 weak promoter W (5′-ACTTTGGACATCCG TCAAGT-3′) were incubated with 100 nM KP34 RNAP to direct to the transcription of their downstream sequence that encodes the same 37 nt RNA. [α-32P]ATP was added into reactions for imaging and visualization. Reaction products were separated by a 25% TBE-Urea denaturing gel. (D) Identification of the full KP34 strong promoter. A KP34 strong promoter S1 (5′-TAATGTTACAGGAGTA-3′) or the 3′ 14 nt common sequence of the two KP34 strong promoters (5′-ATGTTA CAGGAGTA-3′) was inserted into plasmid pUC19 to direct the transcription of their downstream sequences, respectively. A run-off transcript of ∼2700 nt and a terminated transcript of ∼1000 nt (terminated by a predictable T7 class I hairpin terminator structure) were expected from the linearized form of these plasmids if the inserted promoter is sufficient to direct transcription by KP34 RNAP. M: ssRNA Ladder. (E) KP34 promoters in the genome (location of strong promoter (S) pointed by solid arrow and weak promoter (W) by dotted arrow) and comparison of typical ssRNAP promoters. Conserved sequence among ssRNAP promoters are in bold and those homologous between Syn5 promoter and KP34 weak promoter are underlined.

    Techniques Used: In Vitro, Agarose Gel Electrophoresis, Sequencing, Incubation, Imaging, Plasmid Preparation

    Synthesis of a 50 nt RNA containing 3′ hairpin structure by various RNAPs. (A) The 50 nt RNA sequence is shown at the top of the gel. The three DNA templates containing the same coding sequences for the 50 nt run-off RNA transcripts under the control of either a T7 promoter, a KP34 strong promoter, or a Syn5 promoter were incubated with 0.2 μM T7 RNAP, 1 μM KP34 RNAP, or 1 μM Syn5 RNAP, respectively. Incubation with KP34 and T7 RNAP was at 37°C for 1 h and incubation with Syn5 RNAP was at 24°C for 1 h. Reaction products were separated by a 12% TBE native gel and then stained with ethidium bromide. M: ssRNA Ladder. (B) RNA-Seq analysis of the 3′ termini of T7 RNAP transcripts. 3′ termini of sequences with reads more than 1% of total reads were aligned and shown. Percentage of major sequences in total sequencing results are noted and percentage of the correct product is in bold. A dotted line cut indicates the precise terminus encoded by DNA template, and the number of extended nt is shown as n + x. Bold sequences indicate complementary sequences in each RNA specie resulted from extension of a possible 3′ self-primed structure. (C) Similar as B, RNA-Seq analysis of the 3′ termini of KP34 RNAP transcripts. Number of missing nt at the 3′ terminus of major sequences is shown as n–x.
    Figure Legend Snippet: Synthesis of a 50 nt RNA containing 3′ hairpin structure by various RNAPs. (A) The 50 nt RNA sequence is shown at the top of the gel. The three DNA templates containing the same coding sequences for the 50 nt run-off RNA transcripts under the control of either a T7 promoter, a KP34 strong promoter, or a Syn5 promoter were incubated with 0.2 μM T7 RNAP, 1 μM KP34 RNAP, or 1 μM Syn5 RNAP, respectively. Incubation with KP34 and T7 RNAP was at 37°C for 1 h and incubation with Syn5 RNAP was at 24°C for 1 h. Reaction products were separated by a 12% TBE native gel and then stained with ethidium bromide. M: ssRNA Ladder. (B) RNA-Seq analysis of the 3′ termini of T7 RNAP transcripts. 3′ termini of sequences with reads more than 1% of total reads were aligned and shown. Percentage of major sequences in total sequencing results are noted and percentage of the correct product is in bold. A dotted line cut indicates the precise terminus encoded by DNA template, and the number of extended nt is shown as n + x. Bold sequences indicate complementary sequences in each RNA specie resulted from extension of a possible 3′ self-primed structure. (C) Similar as B, RNA-Seq analysis of the 3′ termini of KP34 RNAP transcripts. Number of missing nt at the 3′ terminus of major sequences is shown as n–x.

    Techniques Used: Sequencing, Incubation, Staining, RNA Sequencing Assay

    Synthesis of an sgRNA by T7 and KP34 RNAP. (A) The sgRNA sequence is shown at the top of the gel. The three DNA templates containing the same coding sequences for the sgRNA under the control of either a T7 promoter, a KP34 strong promoter, or a Syn5 promoter were incubated with 0.2 μM T7 RNAP, 1 μM KP34 RNAP, or 1 μM Syn5 RNAP, respectively. Incubation with KP34 and T7 RNAP was at 37°C for 1 h and incubation with Syn5 RNAP was at 24°C for 1 h. Reaction products were separated by a 12% TBE native gel and then stained with ethidium bromide. M: ssRNA Ladder. (B) 3′-RACE analysis of the 3′ termini of T7 RNAP transcripts. 3′ termini of obtained sequences were aligned and shown. A dotted line cut indicates the precise terminus encoded by DNA template and number of extended or missing nt is shown as n + x or n–x. Bold sequences indicate complementary sequences in each RNA resulted from extension of possible 3′ self-primed structures. (C) Similar as B, 3′-RACE analysis of the 3′ termini of KP34 RNAP transcripts.
    Figure Legend Snippet: Synthesis of an sgRNA by T7 and KP34 RNAP. (A) The sgRNA sequence is shown at the top of the gel. The three DNA templates containing the same coding sequences for the sgRNA under the control of either a T7 promoter, a KP34 strong promoter, or a Syn5 promoter were incubated with 0.2 μM T7 RNAP, 1 μM KP34 RNAP, or 1 μM Syn5 RNAP, respectively. Incubation with KP34 and T7 RNAP was at 37°C for 1 h and incubation with Syn5 RNAP was at 24°C for 1 h. Reaction products were separated by a 12% TBE native gel and then stained with ethidium bromide. M: ssRNA Ladder. (B) 3′-RACE analysis of the 3′ termini of T7 RNAP transcripts. 3′ termini of obtained sequences were aligned and shown. A dotted line cut indicates the precise terminus encoded by DNA template and number of extended or missing nt is shown as n + x or n–x. Bold sequences indicate complementary sequences in each RNA resulted from extension of possible 3′ self-primed structures. (C) Similar as B, 3′-RACE analysis of the 3′ termini of KP34 RNAP transcripts.

    Techniques Used: Sequencing, Incubation, Staining

    7) Product Images from "In vitro transcription using psychrophilic phage VSW-3 RNA polymerase"

    Article Title: In vitro transcription using psychrophilic phage VSW-3 RNA polymerase

    Journal: bioRxiv

    doi: 10.1101/2020.09.14.297226

    Response of ssRNAPs to Class II terminator. (A) Using PCR-amplified templates for cas9-RNA IVT, obvious abortive RNA transcripts were observed for T7 RNAP and Syn5 RNAP but not VSW-3 RNAP (top gel). 3’-RACE revealed that the T7 RNAP transcription was terminated 9 nt downstream of a Class II terminator “ATCTGTT” (bottom sequencing result). (B) VSW-3 RNAP IVT was not terminated (no additional bands comparing lane 2 with lane 1) when a Class II terminator “ATCTGTT” was inserted into the middle of the copGFP RNA coding sequence.
    Figure Legend Snippet: Response of ssRNAPs to Class II terminator. (A) Using PCR-amplified templates for cas9-RNA IVT, obvious abortive RNA transcripts were observed for T7 RNAP and Syn5 RNAP but not VSW-3 RNAP (top gel). 3’-RACE revealed that the T7 RNAP transcription was terminated 9 nt downstream of a Class II terminator “ATCTGTT” (bottom sequencing result). (B) VSW-3 RNAP IVT was not terminated (no additional bands comparing lane 2 with lane 1) when a Class II terminator “ATCTGTT” was inserted into the middle of the copGFP RNA coding sequence.

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing

    RNA 3’ extension and RdRp activity of T7 and VSW-3 RNAP. (A) The secondary structure of a sgRNA predicted with RNAfold software. (B) IVT synthesis of a sgRNA (targeting eGFP) by VSW-3 and T7 RNAP. (C) 3’-RACE of the sgRNAs transcripts from T7 and VSW-3 RNAP IVT. Only the 3’ region (red sequence on the top) of the full sgRNA in sequencing results was shown. The length of each sequence was noted. The sequences matching the exact run-off sgRNA (103 nt) was indicated by red stars. (D) Schematic showing the mechanism and origin (3’ self-templated extension by the RdRp activity of T7 RNAP) of the 16 nt 3’-extension in T7 RNAP products as in (C) . (E) T7 but not VSW-3 RNAP retains the RdRp activity to extend purified sgRNA (with terminal primer/template structure).
    Figure Legend Snippet: RNA 3’ extension and RdRp activity of T7 and VSW-3 RNAP. (A) The secondary structure of a sgRNA predicted with RNAfold software. (B) IVT synthesis of a sgRNA (targeting eGFP) by VSW-3 and T7 RNAP. (C) 3’-RACE of the sgRNAs transcripts from T7 and VSW-3 RNAP IVT. Only the 3’ region (red sequence on the top) of the full sgRNA in sequencing results was shown. The length of each sequence was noted. The sequences matching the exact run-off sgRNA (103 nt) was indicated by red stars. (D) Schematic showing the mechanism and origin (3’ self-templated extension by the RdRp activity of T7 RNAP) of the 16 nt 3’-extension in T7 RNAP products as in (C) . (E) T7 but not VSW-3 RNAP retains the RdRp activity to extend purified sgRNA (with terminal primer/template structure).

    Techniques Used: Activity Assay, Software, Sequencing, Purification

    VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized pUC19 plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The RNA yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.
    Figure Legend Snippet: VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized pUC19 plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The RNA yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.

    Techniques Used: SDS Page, Purification, Staining, Plasmid Preparation, Sequencing

    dsRNA by-products from T7 and VSW-3 RNAP IVT. (A) DNA templates for the IVT synthesis of various RNA as indicated on top of the gel. For each RNA, there are two DNA templates differ only in the promoter region to serve for VSW-3 and T7 RNAP IVT, respectively. DNA concentration and purity were compared in 1.5% agarose gel stained with ethidium bromide. (B) After template DNA was removed by DNase I treatment and purified with Monarch RNA Cleanup kit, 1μg of sox7, tdTomato, copGFP and Cas9 RNA transcribed by VSW-3 RNAP and T7 RNAP were analyzed in 1.5% agarose gel stained with ethidium bromide. The white and black colors for bands and background were converted in this gel picture to make the weak double-stranded and abortive RNA bands clearer. (C) Dot blot analysis of the RNA products (each 200 ng) as in (B) by VSW-3 RNAP and T7 RNAP with J2 monoclonal antibody. A prepared dsRNA (351 bp) was applied as quantitative standard (0.1 ng, 0.25 ng, 0.5 ng, 1.0 ng). (D) The gray value measurement and calculation of the X film image (top image in (C) ) by Image J software demonstrating the level of dsRNA contamination in T7 and VSW-3 RNAP transcripts.
    Figure Legend Snippet: dsRNA by-products from T7 and VSW-3 RNAP IVT. (A) DNA templates for the IVT synthesis of various RNA as indicated on top of the gel. For each RNA, there are two DNA templates differ only in the promoter region to serve for VSW-3 and T7 RNAP IVT, respectively. DNA concentration and purity were compared in 1.5% agarose gel stained with ethidium bromide. (B) After template DNA was removed by DNase I treatment and purified with Monarch RNA Cleanup kit, 1μg of sox7, tdTomato, copGFP and Cas9 RNA transcribed by VSW-3 RNAP and T7 RNAP were analyzed in 1.5% agarose gel stained with ethidium bromide. The white and black colors for bands and background were converted in this gel picture to make the weak double-stranded and abortive RNA bands clearer. (C) Dot blot analysis of the RNA products (each 200 ng) as in (B) by VSW-3 RNAP and T7 RNAP with J2 monoclonal antibody. A prepared dsRNA (351 bp) was applied as quantitative standard (0.1 ng, 0.25 ng, 0.5 ng, 1.0 ng). (D) The gray value measurement and calculation of the X film image (top image in (C) ) by Image J software demonstrating the level of dsRNA contamination in T7 and VSW-3 RNAP transcripts.

    Techniques Used: Concentration Assay, Agarose Gel Electrophoresis, Staining, Purification, Dot Blot, Software

    8) Product Images from "Active coacervate droplets as a model for membraneless organelles and protocells"

    Article Title: Active coacervate droplets as a model for membraneless organelles and protocells

    Journal: Nature Communications

    doi: 10.1038/s41467-020-18815-9

    Functional RNA inside fuel-driven droplets. a Schematic representation of the experimental procedure for dynamic droplets with functional RNA. b Confocal micrographs of 23 mM precursor, 4.1 mM poly-U, 25 mM EDC with 0.2 µM Cy5-RNA (SunY ribozyme, Hammerhead ribozyme or Broccoli aptamer), 5 min after addition of EDC. c Confocal micrographs of the SunY containing solution described in d (with 27 mM EDC) at different time points before or after EDC addition. d Fluorescence intensity of solutions containing the Broccoli aptamer with or without DFHB1T (ligand), in the presence and absence of droplets. Maximum fluorescence intensity at 504 nm. Standard conditions with 2 mM MgCl 2 , 30 mM KCl, and 1.5 µM Broccoli aptamer. Addition of 10 mM EDC (fuel) to induce droplet formation. e Confocal and bright field micrographs under the conditions described in d , 5 min after the addition of 15 mM EDC. Experiments were perfomed for n = 2. Source data are provided as a Source Data file.
    Figure Legend Snippet: Functional RNA inside fuel-driven droplets. a Schematic representation of the experimental procedure for dynamic droplets with functional RNA. b Confocal micrographs of 23 mM precursor, 4.1 mM poly-U, 25 mM EDC with 0.2 µM Cy5-RNA (SunY ribozyme, Hammerhead ribozyme or Broccoli aptamer), 5 min after addition of EDC. c Confocal micrographs of the SunY containing solution described in d (with 27 mM EDC) at different time points before or after EDC addition. d Fluorescence intensity of solutions containing the Broccoli aptamer with or without DFHB1T (ligand), in the presence and absence of droplets. Maximum fluorescence intensity at 504 nm. Standard conditions with 2 mM MgCl 2 , 30 mM KCl, and 1.5 µM Broccoli aptamer. Addition of 10 mM EDC (fuel) to induce droplet formation. e Confocal and bright field micrographs under the conditions described in d , 5 min after the addition of 15 mM EDC. Experiments were perfomed for n = 2. Source data are provided as a Source Data file.

    Techniques Used: Functional Assay, Fluorescence

    9) Product Images from "In vitro transcription using psychrophilic phage VSW-3 RNA polymerase"

    Article Title: In vitro transcription using psychrophilic phage VSW-3 RNA polymerase

    Journal: bioRxiv

    doi: 10.1101/2020.09.14.297226

    Optimal VSW-3 RNAP IVT conditions. (A) Screening for the optimal Mg 2+ /NTP concentration in the presence of 1 mM DTT. RNA yield with various optimal Mg 2+ /NTP concentration combination was further compared (gel in the dotted box). The stability of the optimal VSW-3 RNAP IVT buffer with 1 mM DTT was examined (gel in the solid box). (B) Screening for the optimal DTT/ Mg 2+ concentration for the stable and high-yield VSW-3 RNAP IVT buffer. The stability of the high-yield VSW-3 RNAP IVT buffer containing 16 mM Mg 2+ , 4 mM NTP and 5 mM DTT was examined (gel in the solid box). (C) The optimal reaction temperature of VSW-3 RNAP (25°C) for maximum run-off RNA yield. (D) The optimal enzyme concentration of VSW-3 RNAP (0.15 μM) for maximum run-off RNA yield. (E) Optimal IVT yield of VSW-3 RNAP with various reaction temperature/incubation time combinations. The maximum run-off RNA yield was obtained at 25°C for 12 hours. (F) Gray-scale quantitation of the run-off RNA transcripts in gel (E) . Diagram was made using GraphPad Prism. In all gels the bands corresponding to DNA templates were indicated by empty stars and the bands corresponding to run-off RNA transcripts were indicated by filled stars.
    Figure Legend Snippet: Optimal VSW-3 RNAP IVT conditions. (A) Screening for the optimal Mg 2+ /NTP concentration in the presence of 1 mM DTT. RNA yield with various optimal Mg 2+ /NTP concentration combination was further compared (gel in the dotted box). The stability of the optimal VSW-3 RNAP IVT buffer with 1 mM DTT was examined (gel in the solid box). (B) Screening for the optimal DTT/ Mg 2+ concentration for the stable and high-yield VSW-3 RNAP IVT buffer. The stability of the high-yield VSW-3 RNAP IVT buffer containing 16 mM Mg 2+ , 4 mM NTP and 5 mM DTT was examined (gel in the solid box). (C) The optimal reaction temperature of VSW-3 RNAP (25°C) for maximum run-off RNA yield. (D) The optimal enzyme concentration of VSW-3 RNAP (0.15 μM) for maximum run-off RNA yield. (E) Optimal IVT yield of VSW-3 RNAP with various reaction temperature/incubation time combinations. The maximum run-off RNA yield was obtained at 25°C for 12 hours. (F) Gray-scale quantitation of the run-off RNA transcripts in gel (E) . Diagram was made using GraphPad Prism. In all gels the bands corresponding to DNA templates were indicated by empty stars and the bands corresponding to run-off RNA transcripts were indicated by filled stars.

    Techniques Used: Concentration Assay, Incubation, Quantitation Assay

    VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized pUC19 plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The RNA yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.
    Figure Legend Snippet: VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized pUC19 plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The RNA yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.

    Techniques Used: SDS Page, Purification, Staining, Plasmid Preparation, Sequencing

    dsRNA by-products from T7 and VSW-3 RNAP IVT. (A) DNA templates for the IVT synthesis of various RNA as indicated on top of the gel. For each RNA, there are two DNA templates differ only in the promoter region to serve for VSW-3 and T7 RNAP IVT, respectively. DNA concentration and purity were compared in 1.5% agarose gel stained with ethidium bromide. (B) After template DNA was removed by DNase I treatment and purified with Monarch RNA Cleanup kit, 1μg of sox7, tdTomato, copGFP and Cas9 RNA transcribed by VSW-3 RNAP and T7 RNAP were analyzed in 1.5% agarose gel stained with ethidium bromide. The white and black colors for bands and background were converted in this gel picture to make the weak double-stranded and abortive RNA bands clearer. (C) Dot blot analysis of the RNA products (each 200 ng) as in (B) by VSW-3 RNAP and T7 RNAP with J2 monoclonal antibody. A prepared dsRNA (351 bp) was applied as quantitative standard (0.1 ng, 0.25 ng, 0.5 ng, 1.0 ng). (D) The gray value measurement and calculation of the X film image (top image in (C) ) by Image J software demonstrating the level of dsRNA contamination in T7 and VSW-3 RNAP transcripts.
    Figure Legend Snippet: dsRNA by-products from T7 and VSW-3 RNAP IVT. (A) DNA templates for the IVT synthesis of various RNA as indicated on top of the gel. For each RNA, there are two DNA templates differ only in the promoter region to serve for VSW-3 and T7 RNAP IVT, respectively. DNA concentration and purity were compared in 1.5% agarose gel stained with ethidium bromide. (B) After template DNA was removed by DNase I treatment and purified with Monarch RNA Cleanup kit, 1μg of sox7, tdTomato, copGFP and Cas9 RNA transcribed by VSW-3 RNAP and T7 RNAP were analyzed in 1.5% agarose gel stained with ethidium bromide. The white and black colors for bands and background were converted in this gel picture to make the weak double-stranded and abortive RNA bands clearer. (C) Dot blot analysis of the RNA products (each 200 ng) as in (B) by VSW-3 RNAP and T7 RNAP with J2 monoclonal antibody. A prepared dsRNA (351 bp) was applied as quantitative standard (0.1 ng, 0.25 ng, 0.5 ng, 1.0 ng). (D) The gray value measurement and calculation of the X film image (top image in (C) ) by Image J software demonstrating the level of dsRNA contamination in T7 and VSW-3 RNAP transcripts.

    Techniques Used: Concentration Assay, Agarose Gel Electrophoresis, Staining, Purification, Dot Blot, Software

    Related Articles

    Synthesized:

    Article Title: In vitro transcription using psychrophilic phage VSW-3 RNA polymerase
    Article Snippet: 10 colonies from each group were picked for Sanger sequencing. .. In addition, the sgRNA products synthesized by VSW-3 RNAP and T7 RNAP were purified with Monarch RNA Cleanup kit (New England Biolabs) and then further purified by high performance liquid chromatography (HPLC) using PLRP-S column (4000 A, 8 μM, 4.6 × 150 mm). .. In order to prove that the origin of the sgRNA 3’ extension is from the RNA-dependent RNA polymerase (RdRp) activity of T7 RNAP , the HPLC purified VSW-3 sgRNA products were applied as the RdRp template for T7 RNAP and VSW-3 RNAP (final sgRNA concentration: 0.5 μg/μl), other components in the reaction included: 40 mM Tris-HCl pH 8.0, 16 mM MgCl2, 5 mM DTT, 2 mM spermidine, 4 mM each of the four NTPs, 0.2 μM inorganic pyrophosphatase, 1.5 U RNase inhibitor and 0.15 μM T7 RNAP (37°C for 1 hour) or VSW-3 RNAP (25°C for 12 hours).

    Purification:

    Article Title: In vitro transcription using psychrophilic phage VSW-3 RNA polymerase
    Article Snippet: 10 colonies from each group were picked for Sanger sequencing. .. In addition, the sgRNA products synthesized by VSW-3 RNAP and T7 RNAP were purified with Monarch RNA Cleanup kit (New England Biolabs) and then further purified by high performance liquid chromatography (HPLC) using PLRP-S column (4000 A, 8 μM, 4.6 × 150 mm). .. In order to prove that the origin of the sgRNA 3’ extension is from the RNA-dependent RNA polymerase (RdRp) activity of T7 RNAP , the HPLC purified VSW-3 sgRNA products were applied as the RdRp template for T7 RNAP and VSW-3 RNAP (final sgRNA concentration: 0.5 μg/μl), other components in the reaction included: 40 mM Tris-HCl pH 8.0, 16 mM MgCl2, 5 mM DTT, 2 mM spermidine, 4 mM each of the four NTPs, 0.2 μM inorganic pyrophosphatase, 1.5 U RNase inhibitor and 0.15 μM T7 RNAP (37°C for 1 hour) or VSW-3 RNAP (25°C for 12 hours).

    Article Title: Active coacervate droplets as a model for membraneless organelles and protocells
    Article Snippet: RNAs were synthesized by run-off transcription using the MEGAshortscript T7 Transcription Kit (Thermo Scientific). .. After column purification (Monarch RNA Cleanup Kit, NEB), RNAs were tagged with Cy5 fluorophore by ligating a RNA-pentamer modified with a 5′ phosphate and 3′ Cy5 using T4 RNA ligase 2 in the presence complementary DNA splint to the 3′-terminal 15 nucleotides of the RNA product. .. The ligated RNAs were then gel-purified after PAGE.

    Article Title: In vitro transcription using psychrophilic phage VSW-3 RNA polymerase
    Article Snippet: Then 1 μl DNase I was added into reaction mixture and incubation was extended for 30 min at 37°C to remove template DNA. .. The transcripts (pUC19-RNA) were then purified with Monarch RNA Cleanup kit. .. 5’-RACE of the pUC19-RNA from above step began with the RNA 5’ mono-phosphorylation treatment using the Apyrase according to New England Biolabs manual.

    Article Title: An RNA polymerase ribozyme that synthesizes its own ancestor
    Article Snippet: The extension products were eluted from the beads by incubation in 95% formamide and 10 mM EDTA at 95 °C for 10 min, then collected using a centrifugal filter. .. Free streptavidin monomers were removed using the Monarch RNA Cleanup Kit (New England BioLabs), then the full-length products were separated by PAGE, eluted from the gel, purified by ethanol precipitation, and quantified by comparing their fluorescence intensity to known standards by analytical PAGE. .. Purified fragment 1 RNAs were ligated to the Universal miRNA Cloning Linker as described above, then reverse-transcribed using primer Rev3.

    Article Title: Klebsiella Phage KP34 RNA Polymerase and Its Use in RNA Synthesis
    Article Snippet: After electrophoresis, gels were dried and the radioactivity was analyzed using a Fuji BAS 1000 Bioimaging Analyzer. .. RNA-Seq In vitro transcription products were treated with DNase I for 20 min at 37°C to remove the DNA templates and then were purified with Monarch® RNA Cleanup Kit (New England BioLabs). .. Transcripts were incubated with RNA 5′ Pyrophosphohydrolase (RppH) at 37°C for 30 min to remove pyrophosphate from the 5′ end of triphosphorylated RNAs and to leave 5′ monophosphate.

    High Performance Liquid Chromatography:

    Article Title: In vitro transcription using psychrophilic phage VSW-3 RNA polymerase
    Article Snippet: 10 colonies from each group were picked for Sanger sequencing. .. In addition, the sgRNA products synthesized by VSW-3 RNAP and T7 RNAP were purified with Monarch RNA Cleanup kit (New England Biolabs) and then further purified by high performance liquid chromatography (HPLC) using PLRP-S column (4000 A, 8 μM, 4.6 × 150 mm). .. In order to prove that the origin of the sgRNA 3’ extension is from the RNA-dependent RNA polymerase (RdRp) activity of T7 RNAP , the HPLC purified VSW-3 sgRNA products were applied as the RdRp template for T7 RNAP and VSW-3 RNAP (final sgRNA concentration: 0.5 μg/μl), other components in the reaction included: 40 mM Tris-HCl pH 8.0, 16 mM MgCl2, 5 mM DTT, 2 mM spermidine, 4 mM each of the four NTPs, 0.2 μM inorganic pyrophosphatase, 1.5 U RNase inhibitor and 0.15 μM T7 RNAP (37°C for 1 hour) or VSW-3 RNAP (25°C for 12 hours).

    Modification:

    Article Title: Active coacervate droplets as a model for membraneless organelles and protocells
    Article Snippet: RNAs were synthesized by run-off transcription using the MEGAshortscript T7 Transcription Kit (Thermo Scientific). .. After column purification (Monarch RNA Cleanup Kit, NEB), RNAs were tagged with Cy5 fluorophore by ligating a RNA-pentamer modified with a 5′ phosphate and 3′ Cy5 using T4 RNA ligase 2 in the presence complementary DNA splint to the 3′-terminal 15 nucleotides of the RNA product. .. The ligated RNAs were then gel-purified after PAGE.

    Recombinant:

    Article Title: In vitro transcription using psychrophilic phage VSW-3 RNA polymerase
    Article Snippet: Preparative Superdex S200 for gel filtration was from GE Healthcare. .. The Gibson assembly kit, T4 RNA ligase I, Recombinant inorganic pyrophosphatase, rNTPs, DNase I, Apyrase, T7 RNA polymerase (50 U/μl), Low Range ssRNA Ladder and Monarch RNA Cleanup kit were from New England Biolabs. .. 2’-fluoro-dNTPs were from TriLink BioTechnologies.

    Polyacrylamide Gel Electrophoresis:

    Article Title: An RNA polymerase ribozyme that synthesizes its own ancestor
    Article Snippet: The extension products were eluted from the beads by incubation in 95% formamide and 10 mM EDTA at 95 °C for 10 min, then collected using a centrifugal filter. .. Free streptavidin monomers were removed using the Monarch RNA Cleanup Kit (New England BioLabs), then the full-length products were separated by PAGE, eluted from the gel, purified by ethanol precipitation, and quantified by comparing their fluorescence intensity to known standards by analytical PAGE. .. Purified fragment 1 RNAs were ligated to the Universal miRNA Cloning Linker as described above, then reverse-transcribed using primer Rev3.

    Ethanol Precipitation:

    Article Title: An RNA polymerase ribozyme that synthesizes its own ancestor
    Article Snippet: The extension products were eluted from the beads by incubation in 95% formamide and 10 mM EDTA at 95 °C for 10 min, then collected using a centrifugal filter. .. Free streptavidin monomers were removed using the Monarch RNA Cleanup Kit (New England BioLabs), then the full-length products were separated by PAGE, eluted from the gel, purified by ethanol precipitation, and quantified by comparing their fluorescence intensity to known standards by analytical PAGE. .. Purified fragment 1 RNAs were ligated to the Universal miRNA Cloning Linker as described above, then reverse-transcribed using primer Rev3.

    Fluorescence:

    Article Title: An RNA polymerase ribozyme that synthesizes its own ancestor
    Article Snippet: The extension products were eluted from the beads by incubation in 95% formamide and 10 mM EDTA at 95 °C for 10 min, then collected using a centrifugal filter. .. Free streptavidin monomers were removed using the Monarch RNA Cleanup Kit (New England BioLabs), then the full-length products were separated by PAGE, eluted from the gel, purified by ethanol precipitation, and quantified by comparing their fluorescence intensity to known standards by analytical PAGE. .. Purified fragment 1 RNAs were ligated to the Universal miRNA Cloning Linker as described above, then reverse-transcribed using primer Rev3.

    RNA Sequencing Assay:

    Article Title: Klebsiella Phage KP34 RNA Polymerase and Its Use in RNA Synthesis
    Article Snippet: After electrophoresis, gels were dried and the radioactivity was analyzed using a Fuji BAS 1000 Bioimaging Analyzer. .. RNA-Seq In vitro transcription products were treated with DNase I for 20 min at 37°C to remove the DNA templates and then were purified with Monarch® RNA Cleanup Kit (New England BioLabs). .. Transcripts were incubated with RNA 5′ Pyrophosphohydrolase (RppH) at 37°C for 30 min to remove pyrophosphate from the 5′ end of triphosphorylated RNAs and to leave 5′ monophosphate.

    In Vitro:

    Article Title: Klebsiella Phage KP34 RNA Polymerase and Its Use in RNA Synthesis
    Article Snippet: After electrophoresis, gels were dried and the radioactivity was analyzed using a Fuji BAS 1000 Bioimaging Analyzer. .. RNA-Seq In vitro transcription products were treated with DNase I for 20 min at 37°C to remove the DNA templates and then were purified with Monarch® RNA Cleanup Kit (New England BioLabs). .. Transcripts were incubated with RNA 5′ Pyrophosphohydrolase (RppH) at 37°C for 30 min to remove pyrophosphate from the 5′ end of triphosphorylated RNAs and to leave 5′ monophosphate.

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  • 98
    New England Biolabs monarch rna cleanup kit
    VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized <t>pUC19</t> plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The <t>RNA</t> yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.
    Monarch Rna Cleanup Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monarch rna cleanup kit/product/New England Biolabs
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    monarch rna cleanup kit - by Bioz Stars, 2021-04
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    98
    New England Biolabs monarch rna prep kit
    Diverse Transcription-Regulatory Control of Stag1 in ES cells. (A, B) 5’ Rapid Amplification of cDNA ends (RACE) for SA1 in ES and EpiLC cells. Arrows indicate bands which were cloned and sequenced. In A, red star indicates SATS TSS and red arrow indicates canonical TSS. In B: red indicates full length Stag1 with both SATS and can TSSs; dark blue indicates alternatively spliced variants, skipping of various exons in 5’ region; light blue indicates the TSSs at e6, e7. (C) 3’ RACE for SA1 in ES cells. Arrows indicate bands which were cloned and sequenced. Red indicates canonical full-length end; green indicates end in i25. (D) Top, schematic of the Stag1 gene annotation in mm10 and the identified TSS and TTSs from RACE indicated. Bottom, aligned sequence clones from the <t>PCR</t> mini-screen and their predicted impact on the SA1 protein, right. Green arrows and red bars within the transcripts indicate start of the coding sequence and the TTS respectively. Shown also are the regions which code for the AT hook and SCD domains. (E) Percent Spliced In (PSI) calculations based on VAST-Tools analysis of <t>RNA-seq</t> from multiple 2i (blue) and FCS (red) datasets (see Methods for details of libraries). Data are shown relative to Neural Stem cell (NSC) frequencies to highlight the events that are ES-specific. (F) Genome topology at the Stag1 locus. Hi-C contact maps in ES (2i) and NS cells of the 900kb region on chromosome 9 containing the Stag1 topologically associated domain (TAD). TADs are denoted with a vertical line and as repressed (orange) or active (blue). Shown also are tracks for Genes, Nanog and CTCF ChIP-seq as well as a track indicating the directionality of CTCF binding sites (red, forward; blue, reverse). Aligned to the Gene track are also the SA1 transcripts discovered above where red represents the untranslated regions and blue the coding body. UMI-4C-seq viewpoints (asterisks on the ChIP tracks) are positioned to the leftmost CTCF site (‘CTCF bait’) and to the Nanog site 40 kb upstream of the Stag1 canonical TSS (‘Nanog bait’). For each bait, UMI information for each cell type is shown as well as the comparative plots where red represents an enrichment of contacts in ES compared to NS. (G) Top, cartoon depicting functional domains within Stag1 protein, including the AT-hook (aa 3-58); Stromalin conserved domain (SCD, aa 296-381) and the C-terminus. Bottom, the predicted Stag1 protein isoforms based on transcript analysis with estimated sizes for each isoform. Purple boxes in the 105kDa and 90kDa isoforms represent retained introns. (H) PONDR tracks as before shown for the N-terminal truncated (top) and C-terminal truncated (bottom) transcripts. (I) Chromatin immunoprecipitation of SA1 from ES cells. (J) WB analysis of SA1 isoforms in chromatin fractions from ES cells treated with siscr and siSA1. H3 serves as a fraction and loading control. (K) Chromatin immunoprecipitation for the v5 tag in SA1 NG-FKBP ES cells treated with DMSO-only or dTAG. Note, SA1 bands now run 42kDa higher due to the addition of the tag. See also Figure S3, Table S1 and Table S2.
    Monarch Rna Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monarch rna prep kit/product/New England Biolabs
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    monarch rna prep kit - by Bioz Stars, 2021-04
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      Buy from Supplier

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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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    VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized pUC19 plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The RNA yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.

    Journal: bioRxiv

    Article Title: In vitro transcription using psychrophilic phage VSW-3 RNA polymerase

    doi: 10.1101/2020.09.14.297226

    Figure Lengend Snippet: VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized pUC19 plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The RNA yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.

    Article Snippet: The transcripts (pUC19-RNA) were then purified with Monarch RNA Cleanup kit.

    Techniques: SDS Page, Purification, Staining, Plasmid Preparation, Sequencing

    Template-encoded Poly(A) tailing reduces antisense by-product formation. A) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30 bp, 60 bp, 120 bp) of poly-T sequence at 3’-end under standard conditions (5 mM rNTPs, 37°C for 1 hour). B) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly-T (60 bp and 120 bp) sequence at the 3’-end. IVT reactions were performed at 37°C or 50°C.

    Journal: bioRxiv

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    doi: 10.1101/815092

    Figure Lengend Snippet: Template-encoded Poly(A) tailing reduces antisense by-product formation. A) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30 bp, 60 bp, 120 bp) of poly-T sequence at 3’-end under standard conditions (5 mM rNTPs, 37°C for 1 hour). B) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly-T (60 bp and 120 bp) sequence at the 3’-end. IVT reactions were performed at 37°C or 50°C.

    Article Snippet: For the 512B transcripts, reactions were cleaned up with the Monarch® RNA Cleanup Kit (New England Biolabs).

    Techniques: Nucleic Acid Electrophoresis, Synthesized, Sequencing

    Truncation of the 3’-end of the 512B DNA templates results in reduction of the antisense RNA by-product formation. Immunoblot (with J2 antibody; 1:5000; Scicons) and native gel electrophoresis analyses of in vitro transcription reactions performed on 512B template with 3’-end truncations (50 and 200 base pairs). In vitro transcription reactions were performed with TsT7-1 at 37°C or 50°C.

    Journal: bioRxiv

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    doi: 10.1101/815092

    Figure Lengend Snippet: Truncation of the 3’-end of the 512B DNA templates results in reduction of the antisense RNA by-product formation. Immunoblot (with J2 antibody; 1:5000; Scicons) and native gel electrophoresis analyses of in vitro transcription reactions performed on 512B template with 3’-end truncations (50 and 200 base pairs). In vitro transcription reactions were performed with TsT7-1 at 37°C or 50°C.

    Article Snippet: For the 512B transcripts, reactions were cleaned up with the Monarch® RNA Cleanup Kit (New England Biolabs).

    Techniques: Nucleic Acid Electrophoresis, In Vitro

    Optimal VSW-3 RNAP IVT conditions. (A) Screening for the optimal Mg 2+ /NTP concentration in the presence of 1 mM DTT. RNA yield with various optimal Mg 2+ /NTP concentration combination was further compared (gel in the dotted box). The stability of the optimal VSW-3 RNAP IVT buffer with 1 mM DTT was examined (gel in the solid box). (B) Screening for the optimal DTT/ Mg 2+ concentration for the stable and high-yield VSW-3 RNAP IVT buffer. The stability of the high-yield VSW-3 RNAP IVT buffer containing 16 mM Mg 2+ , 4 mM NTP and 5 mM DTT was examined (gel in the solid box). (C) The optimal reaction temperature of VSW-3 RNAP (25°C) for maximum run-off RNA yield. (D) The optimal enzyme concentration of VSW-3 RNAP (0.15 μM) for maximum run-off RNA yield. (E) Optimal IVT yield of VSW-3 RNAP with various reaction temperature/incubation time combinations. The maximum run-off RNA yield was obtained at 25°C for 12 hours. (F) Gray-scale quantitation of the run-off RNA transcripts in gel (E) . Diagram was made using GraphPad Prism. In all gels the bands corresponding to DNA templates were indicated by empty stars and the bands corresponding to run-off RNA transcripts were indicated by filled stars.

    Journal: bioRxiv

    Article Title: In vitro transcription using psychrophilic phage VSW-3 RNA polymerase

    doi: 10.1101/2020.09.14.297226

    Figure Lengend Snippet: Optimal VSW-3 RNAP IVT conditions. (A) Screening for the optimal Mg 2+ /NTP concentration in the presence of 1 mM DTT. RNA yield with various optimal Mg 2+ /NTP concentration combination was further compared (gel in the dotted box). The stability of the optimal VSW-3 RNAP IVT buffer with 1 mM DTT was examined (gel in the solid box). (B) Screening for the optimal DTT/ Mg 2+ concentration for the stable and high-yield VSW-3 RNAP IVT buffer. The stability of the high-yield VSW-3 RNAP IVT buffer containing 16 mM Mg 2+ , 4 mM NTP and 5 mM DTT was examined (gel in the solid box). (C) The optimal reaction temperature of VSW-3 RNAP (25°C) for maximum run-off RNA yield. (D) The optimal enzyme concentration of VSW-3 RNAP (0.15 μM) for maximum run-off RNA yield. (E) Optimal IVT yield of VSW-3 RNAP with various reaction temperature/incubation time combinations. The maximum run-off RNA yield was obtained at 25°C for 12 hours. (F) Gray-scale quantitation of the run-off RNA transcripts in gel (E) . Diagram was made using GraphPad Prism. In all gels the bands corresponding to DNA templates were indicated by empty stars and the bands corresponding to run-off RNA transcripts were indicated by filled stars.

    Article Snippet: In addition, the sgRNA products synthesized by VSW-3 RNAP and T7 RNAP were purified with Monarch RNA Cleanup kit (New England Biolabs) and then further purified by high performance liquid chromatography (HPLC) using PLRP-S column (4000 A, 8 μM, 4.6 × 150 mm).

    Techniques: Concentration Assay, Incubation, Quantitation Assay

    Response of ssRNAPs to Class II terminator. (A) Using PCR-amplified templates for cas9-RNA IVT, obvious abortive RNA transcripts were observed for T7 RNAP and Syn5 RNAP but not VSW-3 RNAP (top gel). 3’-RACE revealed that the T7 RNAP transcription was terminated 9 nt downstream of a Class II terminator “ATCTGTT” (bottom sequencing result). (B) VSW-3 RNAP IVT was not terminated (no additional bands comparing lane 2 with lane 1) when a Class II terminator “ATCTGTT” was inserted into the middle of the copGFP RNA coding sequence.

    Journal: bioRxiv

    Article Title: In vitro transcription using psychrophilic phage VSW-3 RNA polymerase

    doi: 10.1101/2020.09.14.297226

    Figure Lengend Snippet: Response of ssRNAPs to Class II terminator. (A) Using PCR-amplified templates for cas9-RNA IVT, obvious abortive RNA transcripts were observed for T7 RNAP and Syn5 RNAP but not VSW-3 RNAP (top gel). 3’-RACE revealed that the T7 RNAP transcription was terminated 9 nt downstream of a Class II terminator “ATCTGTT” (bottom sequencing result). (B) VSW-3 RNAP IVT was not terminated (no additional bands comparing lane 2 with lane 1) when a Class II terminator “ATCTGTT” was inserted into the middle of the copGFP RNA coding sequence.

    Article Snippet: In addition, the sgRNA products synthesized by VSW-3 RNAP and T7 RNAP were purified with Monarch RNA Cleanup kit (New England Biolabs) and then further purified by high performance liquid chromatography (HPLC) using PLRP-S column (4000 A, 8 μM, 4.6 × 150 mm).

    Techniques: Polymerase Chain Reaction, Amplification, Sequencing

    RNA 3’ extension and RdRp activity of T7 and VSW-3 RNAP. (A) The secondary structure of a sgRNA predicted with RNAfold software. (B) IVT synthesis of a sgRNA (targeting eGFP) by VSW-3 and T7 RNAP. (C) 3’-RACE of the sgRNAs transcripts from T7 and VSW-3 RNAP IVT. Only the 3’ region (red sequence on the top) of the full sgRNA in sequencing results was shown. The length of each sequence was noted. The sequences matching the exact run-off sgRNA (103 nt) was indicated by red stars. (D) Schematic showing the mechanism and origin (3’ self-templated extension by the RdRp activity of T7 RNAP) of the 16 nt 3’-extension in T7 RNAP products as in (C) . (E) T7 but not VSW-3 RNAP retains the RdRp activity to extend purified sgRNA (with terminal primer/template structure).

    Journal: bioRxiv

    Article Title: In vitro transcription using psychrophilic phage VSW-3 RNA polymerase

    doi: 10.1101/2020.09.14.297226

    Figure Lengend Snippet: RNA 3’ extension and RdRp activity of T7 and VSW-3 RNAP. (A) The secondary structure of a sgRNA predicted with RNAfold software. (B) IVT synthesis of a sgRNA (targeting eGFP) by VSW-3 and T7 RNAP. (C) 3’-RACE of the sgRNAs transcripts from T7 and VSW-3 RNAP IVT. Only the 3’ region (red sequence on the top) of the full sgRNA in sequencing results was shown. The length of each sequence was noted. The sequences matching the exact run-off sgRNA (103 nt) was indicated by red stars. (D) Schematic showing the mechanism and origin (3’ self-templated extension by the RdRp activity of T7 RNAP) of the 16 nt 3’-extension in T7 RNAP products as in (C) . (E) T7 but not VSW-3 RNAP retains the RdRp activity to extend purified sgRNA (with terminal primer/template structure).

    Article Snippet: In addition, the sgRNA products synthesized by VSW-3 RNAP and T7 RNAP were purified with Monarch RNA Cleanup kit (New England Biolabs) and then further purified by high performance liquid chromatography (HPLC) using PLRP-S column (4000 A, 8 μM, 4.6 × 150 mm).

    Techniques: Activity Assay, Software, Sequencing, Purification

    dsRNA by-products from T7 and VSW-3 RNAP IVT. (A) DNA templates for the IVT synthesis of various RNA as indicated on top of the gel. For each RNA, there are two DNA templates differ only in the promoter region to serve for VSW-3 and T7 RNAP IVT, respectively. DNA concentration and purity were compared in 1.5% agarose gel stained with ethidium bromide. (B) After template DNA was removed by DNase I treatment and purified with Monarch RNA Cleanup kit, 1μg of sox7, tdTomato, copGFP and Cas9 RNA transcribed by VSW-3 RNAP and T7 RNAP were analyzed in 1.5% agarose gel stained with ethidium bromide. The white and black colors for bands and background were converted in this gel picture to make the weak double-stranded and abortive RNA bands clearer. (C) Dot blot analysis of the RNA products (each 200 ng) as in (B) by VSW-3 RNAP and T7 RNAP with J2 monoclonal antibody. A prepared dsRNA (351 bp) was applied as quantitative standard (0.1 ng, 0.25 ng, 0.5 ng, 1.0 ng). (D) The gray value measurement and calculation of the X film image (top image in (C) ) by Image J software demonstrating the level of dsRNA contamination in T7 and VSW-3 RNAP transcripts.

    Journal: bioRxiv

    Article Title: In vitro transcription using psychrophilic phage VSW-3 RNA polymerase

    doi: 10.1101/2020.09.14.297226

    Figure Lengend Snippet: dsRNA by-products from T7 and VSW-3 RNAP IVT. (A) DNA templates for the IVT synthesis of various RNA as indicated on top of the gel. For each RNA, there are two DNA templates differ only in the promoter region to serve for VSW-3 and T7 RNAP IVT, respectively. DNA concentration and purity were compared in 1.5% agarose gel stained with ethidium bromide. (B) After template DNA was removed by DNase I treatment and purified with Monarch RNA Cleanup kit, 1μg of sox7, tdTomato, copGFP and Cas9 RNA transcribed by VSW-3 RNAP and T7 RNAP were analyzed in 1.5% agarose gel stained with ethidium bromide. The white and black colors for bands and background were converted in this gel picture to make the weak double-stranded and abortive RNA bands clearer. (C) Dot blot analysis of the RNA products (each 200 ng) as in (B) by VSW-3 RNAP and T7 RNAP with J2 monoclonal antibody. A prepared dsRNA (351 bp) was applied as quantitative standard (0.1 ng, 0.25 ng, 0.5 ng, 1.0 ng). (D) The gray value measurement and calculation of the X film image (top image in (C) ) by Image J software demonstrating the level of dsRNA contamination in T7 and VSW-3 RNAP transcripts.

    Article Snippet: In addition, the sgRNA products synthesized by VSW-3 RNAP and T7 RNAP were purified with Monarch RNA Cleanup kit (New England Biolabs) and then further purified by high performance liquid chromatography (HPLC) using PLRP-S column (4000 A, 8 μM, 4.6 × 150 mm).

    Techniques: Concentration Assay, Agarose Gel Electrophoresis, Staining, Purification, Dot Blot, Software

    Diverse Transcription-Regulatory Control of Stag1 in ES cells. (A, B) 5’ Rapid Amplification of cDNA ends (RACE) for SA1 in ES and EpiLC cells. Arrows indicate bands which were cloned and sequenced. In A, red star indicates SATS TSS and red arrow indicates canonical TSS. In B: red indicates full length Stag1 with both SATS and can TSSs; dark blue indicates alternatively spliced variants, skipping of various exons in 5’ region; light blue indicates the TSSs at e6, e7. (C) 3’ RACE for SA1 in ES cells. Arrows indicate bands which were cloned and sequenced. Red indicates canonical full-length end; green indicates end in i25. (D) Top, schematic of the Stag1 gene annotation in mm10 and the identified TSS and TTSs from RACE indicated. Bottom, aligned sequence clones from the PCR mini-screen and their predicted impact on the SA1 protein, right. Green arrows and red bars within the transcripts indicate start of the coding sequence and the TTS respectively. Shown also are the regions which code for the AT hook and SCD domains. (E) Percent Spliced In (PSI) calculations based on VAST-Tools analysis of RNA-seq from multiple 2i (blue) and FCS (red) datasets (see Methods for details of libraries). Data are shown relative to Neural Stem cell (NSC) frequencies to highlight the events that are ES-specific. (F) Genome topology at the Stag1 locus. Hi-C contact maps in ES (2i) and NS cells of the 900kb region on chromosome 9 containing the Stag1 topologically associated domain (TAD). TADs are denoted with a vertical line and as repressed (orange) or active (blue). Shown also are tracks for Genes, Nanog and CTCF ChIP-seq as well as a track indicating the directionality of CTCF binding sites (red, forward; blue, reverse). Aligned to the Gene track are also the SA1 transcripts discovered above where red represents the untranslated regions and blue the coding body. UMI-4C-seq viewpoints (asterisks on the ChIP tracks) are positioned to the leftmost CTCF site (‘CTCF bait’) and to the Nanog site 40 kb upstream of the Stag1 canonical TSS (‘Nanog bait’). For each bait, UMI information for each cell type is shown as well as the comparative plots where red represents an enrichment of contacts in ES compared to NS. (G) Top, cartoon depicting functional domains within Stag1 protein, including the AT-hook (aa 3-58); Stromalin conserved domain (SCD, aa 296-381) and the C-terminus. Bottom, the predicted Stag1 protein isoforms based on transcript analysis with estimated sizes for each isoform. Purple boxes in the 105kDa and 90kDa isoforms represent retained introns. (H) PONDR tracks as before shown for the N-terminal truncated (top) and C-terminal truncated (bottom) transcripts. (I) Chromatin immunoprecipitation of SA1 from ES cells. (J) WB analysis of SA1 isoforms in chromatin fractions from ES cells treated with siscr and siSA1. H3 serves as a fraction and loading control. (K) Chromatin immunoprecipitation for the v5 tag in SA1 NG-FKBP ES cells treated with DMSO-only or dTAG. Note, SA1 bands now run 42kDa higher due to the addition of the tag. See also Figure S3, Table S1 and Table S2.

    Journal: bioRxiv

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

    doi: 10.1101/2021.02.14.429938

    Figure Lengend Snippet: Diverse Transcription-Regulatory Control of Stag1 in ES cells. (A, B) 5’ Rapid Amplification of cDNA ends (RACE) for SA1 in ES and EpiLC cells. Arrows indicate bands which were cloned and sequenced. In A, red star indicates SATS TSS and red arrow indicates canonical TSS. In B: red indicates full length Stag1 with both SATS and can TSSs; dark blue indicates alternatively spliced variants, skipping of various exons in 5’ region; light blue indicates the TSSs at e6, e7. (C) 3’ RACE for SA1 in ES cells. Arrows indicate bands which were cloned and sequenced. Red indicates canonical full-length end; green indicates end in i25. (D) Top, schematic of the Stag1 gene annotation in mm10 and the identified TSS and TTSs from RACE indicated. Bottom, aligned sequence clones from the PCR mini-screen and their predicted impact on the SA1 protein, right. Green arrows and red bars within the transcripts indicate start of the coding sequence and the TTS respectively. Shown also are the regions which code for the AT hook and SCD domains. (E) Percent Spliced In (PSI) calculations based on VAST-Tools analysis of RNA-seq from multiple 2i (blue) and FCS (red) datasets (see Methods for details of libraries). Data are shown relative to Neural Stem cell (NSC) frequencies to highlight the events that are ES-specific. (F) Genome topology at the Stag1 locus. Hi-C contact maps in ES (2i) and NS cells of the 900kb region on chromosome 9 containing the Stag1 topologically associated domain (TAD). TADs are denoted with a vertical line and as repressed (orange) or active (blue). Shown also are tracks for Genes, Nanog and CTCF ChIP-seq as well as a track indicating the directionality of CTCF binding sites (red, forward; blue, reverse). Aligned to the Gene track are also the SA1 transcripts discovered above where red represents the untranslated regions and blue the coding body. UMI-4C-seq viewpoints (asterisks on the ChIP tracks) are positioned to the leftmost CTCF site (‘CTCF bait’) and to the Nanog site 40 kb upstream of the Stag1 canonical TSS (‘Nanog bait’). For each bait, UMI information for each cell type is shown as well as the comparative plots where red represents an enrichment of contacts in ES compared to NS. (G) Top, cartoon depicting functional domains within Stag1 protein, including the AT-hook (aa 3-58); Stromalin conserved domain (SCD, aa 296-381) and the C-terminus. Bottom, the predicted Stag1 protein isoforms based on transcript analysis with estimated sizes for each isoform. Purple boxes in the 105kDa and 90kDa isoforms represent retained introns. (H) PONDR tracks as before shown for the N-terminal truncated (top) and C-terminal truncated (bottom) transcripts. (I) Chromatin immunoprecipitation of SA1 from ES cells. (J) WB analysis of SA1 isoforms in chromatin fractions from ES cells treated with siscr and siSA1. H3 serves as a fraction and loading control. (K) Chromatin immunoprecipitation for the v5 tag in SA1 NG-FKBP ES cells treated with DMSO-only or dTAG. Note, SA1 bands now run 42kDa higher due to the addition of the tag. See also Figure S3, Table S1 and Table S2.

    Article Snippet: qRT-PCR analysis Total RNA was isolated using Monarch RNA prep kit (NEB).

    Techniques: Rapid Amplification of cDNA Ends, Clone Assay, Sequencing, Polymerase Chain Reaction, RNA Sequencing Assay, Hi-C, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, Western Blot