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    New England Biolabs bsu polymerase
    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)
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    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)

    Journal: Nature Communications

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

    doi: 10.1038/s41467-018-02866-0

    Figure Lengend 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)

    Article Snippet: Each reaction component for C1-RamDA-seq was as follows: Lysis Final Mix (1.12 μL of 10% NP40, 4.05 μL RealTime ready Cell Lysis Buffer, 0.84 μL of 40 U/μL RNasin Plus RNase Inhibitor, 3 μL of 1:5000 ERCC RNA Spikes, 1.35 μL of C1 Loading Reagent, and 16.55 μL of RNase-free water), gDNA Digestion Final Mix (2.5 μL of 5× PrimeScript Buffer, 5 μL of 1 U/μL DNase I Amplification Grade, 1 μL of 20× C1 Loading Reagent, and 11.5 μL of RNase-free water), Priming Final Mix (17.23 μL of 5× PrimeScript Buffer, 5.24 μL of PrimeScript RT Enzyme Mix, 0.7 μL of 30 μM oligo(dT)12, 2.8 μL of 100 μM 1st-NSRs, 1.75 μL of 2 mg/mL T4 gene 32 protein (NEB), 1.13 μL of C1 Loading Reagent, and 1.15 μL of RNase-free water), RT Final Mix (12 μL of 5× PrimeScript Buffer, 3 μL of PrimeScript RT Enzyme Mix, RealTime ready Cell Lysis Buffer, 0.4 μL of 30 μM oligo(dT)12, 1.6 μL of 100 μM 1st-NSRs, 1 μL of 2 mg/ml T4 gene 32 protein (NEB), 3.96 μL of 1 U/μL DNase I Amplification Grade, 2.25 μL of C1 Loading Reagent, and 33.92 μL of RNase-free water), Second-strand Final Mix (6.7 μL of 10× NEB buffer 2, 6.7 μL of 2.5 mM each dNTP Mixture, 5.36 μL of 100 μM 2nd-NSRs, 2.01 μL of 5 U/μL Klenow Fragment (3′ → 5′ exo-), 1.5 μL of C1 Loading Reagent, and 7.73 μL of RNase-free water), and Harvest Reagent (500 μL of Tagment DNA Buffer, 237.5 μL of C1 Harvest Reagent, and 12.5 μL of 20× C1 Loading Reagent).

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

    RCA assay in a MOSIC method system. ( A ) Schematic representation of the single-stranded oligonucleotide production by MOSIC method. p378 double-stranded circular nicked DNA (i) is amplified by RCA in two possible ways: single-stranded 378 nt ODN sequence repeated in tandem with hairpin structures in between (ii) and then digested as single-stranded product (iii) or double-stranded DNA repeated in tandem which is digested in double-stranded 378 bp DNA fragments. ( B ) Agarose gel of BseGI digestion products from p378 RCA. RCAs of nicked p378 were stopped at different reaction times from 0.5 to 24 h (lanes 1–14) and the amplifications were performed with (+) and without (−) T4 gene 32 protein. L = 100 bp DNA ladder. The digestion products of RCA performed in absence of T4 gene 32 (odd lanes) correspond to the predicted 378 bp dsDNA which means that phi29 DNA polymerase amplifies p378 mostly in double-stranded form. On the other hand the digestion product of RCA performed with the addition of T4 gene 32 (even lanes) corresponds to the predicted 378 nt ODN as also confirmed from the denaturing PAGE in Supplementary Figure S3.

    Journal: Nucleic Acids Research

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA

    doi: 10.1093/nar/gku737

    Figure Lengend Snippet: RCA assay in a MOSIC method system. ( A ) Schematic representation of the single-stranded oligonucleotide production by MOSIC method. p378 double-stranded circular nicked DNA (i) is amplified by RCA in two possible ways: single-stranded 378 nt ODN sequence repeated in tandem with hairpin structures in between (ii) and then digested as single-stranded product (iii) or double-stranded DNA repeated in tandem which is digested in double-stranded 378 bp DNA fragments. ( B ) Agarose gel of BseGI digestion products from p378 RCA. RCAs of nicked p378 were stopped at different reaction times from 0.5 to 24 h (lanes 1–14) and the amplifications were performed with (+) and without (−) T4 gene 32 protein. L = 100 bp DNA ladder. The digestion products of RCA performed in absence of T4 gene 32 (odd lanes) correspond to the predicted 378 bp dsDNA which means that phi29 DNA polymerase amplifies p378 mostly in double-stranded form. On the other hand the digestion product of RCA performed with the addition of T4 gene 32 (even lanes) corresponds to the predicted 378 nt ODN as also confirmed from the denaturing PAGE in Supplementary Figure S3.

    Article Snippet: We circularized the linear 378-nt pseudogene (5 ng/μl) by T4 ligase (0.25 U/μl; Fermentas) in 1× rapid ligation buffer at 22°C for 10 min, followed by an inactivation step at 65°C for 10 min. We nicked the resulting circular pseudogene (1 ng/μl) by Nb.BsrDI and Nt.BspQI (0.5 U/μl, New England Biolabs) in 1× NEB3 buffer at 65°C for 2 h and we stopped the reaction by heating at 80°C for 20 min. We amplified the nicked circular pseudogene (0.1 ng/μl) at 30°C by RCA and T4 gene 32 (25 ng/μl; NEB) stopping the reaction by heat inactivation (80°C, for 20 min) at different time points.

    Techniques: Amplification, Sequencing, Agarose Gel Electrophoresis, Polyacrylamide Gel Electrophoresis

    RCA assay of pUC19 DNA plasmid. ( A ) Agarose gel of pUC19 RCA products. Lanes 1–7 RCA performed with increasing concentrations of T4 gene 32 protein (0,10, 20, 30, 50, 75, 100 ng/μl respectively); lane 8 negative control with no phi29 DNA polymerase in the reaction mixture; 1 kb plus DNA ladders (L). ( B ) Agarose gel of MlyI digestion test. RCA products in (A) were digested by MlyI restriction enzyme and the corresponding digestion products (9-16) were run on agarose gel. 1 kb plus DNA ladders (L). ( C ) Picogreen assay of pUC19 RCA. The amplification is expressed in percentage of relative fluorescence units (RFU) and the signal of the amplification product without T4 gene 32 is taken as 100%. Both MlyI digestion and picogreen assay confirm that rolling circle amplification makes mostly double-stranded DNA but they also suggest that T4 gene 32 SSB protein drastically reduces dsDNA production.

    Journal: Nucleic Acids Research

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA

    doi: 10.1093/nar/gku737

    Figure Lengend Snippet: RCA assay of pUC19 DNA plasmid. ( A ) Agarose gel of pUC19 RCA products. Lanes 1–7 RCA performed with increasing concentrations of T4 gene 32 protein (0,10, 20, 30, 50, 75, 100 ng/μl respectively); lane 8 negative control with no phi29 DNA polymerase in the reaction mixture; 1 kb plus DNA ladders (L). ( B ) Agarose gel of MlyI digestion test. RCA products in (A) were digested by MlyI restriction enzyme and the corresponding digestion products (9-16) were run on agarose gel. 1 kb plus DNA ladders (L). ( C ) Picogreen assay of pUC19 RCA. The amplification is expressed in percentage of relative fluorescence units (RFU) and the signal of the amplification product without T4 gene 32 is taken as 100%. Both MlyI digestion and picogreen assay confirm that rolling circle amplification makes mostly double-stranded DNA but they also suggest that T4 gene 32 SSB protein drastically reduces dsDNA production.

    Article Snippet: We circularized the linear 378-nt pseudogene (5 ng/μl) by T4 ligase (0.25 U/μl; Fermentas) in 1× rapid ligation buffer at 22°C for 10 min, followed by an inactivation step at 65°C for 10 min. We nicked the resulting circular pseudogene (1 ng/μl) by Nb.BsrDI and Nt.BspQI (0.5 U/μl, New England Biolabs) in 1× NEB3 buffer at 65°C for 2 h and we stopped the reaction by heating at 80°C for 20 min. We amplified the nicked circular pseudogene (0.1 ng/μl) at 30°C by RCA and T4 gene 32 (25 ng/μl; NEB) stopping the reaction by heat inactivation (80°C, for 20 min) at different time points.

    Techniques: Plasmid Preparation, Agarose Gel Electrophoresis, Negative Control, Picogreen Assay, Amplification, Fluorescence

    Single and double-stranded DNA production changes in RCA of p378 circular DNA over time. ( A ) BseGI digested RCA products performed without SSB protein over time (1–72 h) loaded on agarose gel (above) and denaturing polyacrilamide gel (below). ( B ) BseGI digested RCA products performed with SSB protein over time (3–72 h) loaded on agarose gel (above) and denaturing polyacrilamide gel (below). ( C ) Plotted concentrations of single and double-stranded RCA products expressed in nanograms per microliter over time. Single-stranded DNA was measured as deduction of the double-stranded DNA band intensities (agarose gels) from the total DNA band intensities (denaturing polyacrilamide gels). Linearity of the band intensities corresponding to the DNA amount range used for our experimental condition was verified (See Supplementary Figure S4). ( D ) Single and double-stranded DNA in RCA over time expressed as mass fraction. In the initial hours of a rolling circle amplification without SSB protein most of the amplicons are in the single-stranded form but then all the DNA is converted into the double-stranded form. The addition of SSB protein T4 gene 32 drastically reduces the conversion of single-stranded DNA into double-stranded form.

    Journal: Nucleic Acids Research

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA

    doi: 10.1093/nar/gku737

    Figure Lengend Snippet: Single and double-stranded DNA production changes in RCA of p378 circular DNA over time. ( A ) BseGI digested RCA products performed without SSB protein over time (1–72 h) loaded on agarose gel (above) and denaturing polyacrilamide gel (below). ( B ) BseGI digested RCA products performed with SSB protein over time (3–72 h) loaded on agarose gel (above) and denaturing polyacrilamide gel (below). ( C ) Plotted concentrations of single and double-stranded RCA products expressed in nanograms per microliter over time. Single-stranded DNA was measured as deduction of the double-stranded DNA band intensities (agarose gels) from the total DNA band intensities (denaturing polyacrilamide gels). Linearity of the band intensities corresponding to the DNA amount range used for our experimental condition was verified (See Supplementary Figure S4). ( D ) Single and double-stranded DNA in RCA over time expressed as mass fraction. In the initial hours of a rolling circle amplification without SSB protein most of the amplicons are in the single-stranded form but then all the DNA is converted into the double-stranded form. The addition of SSB protein T4 gene 32 drastically reduces the conversion of single-stranded DNA into double-stranded form.

    Article Snippet: We circularized the linear 378-nt pseudogene (5 ng/μl) by T4 ligase (0.25 U/μl; Fermentas) in 1× rapid ligation buffer at 22°C for 10 min, followed by an inactivation step at 65°C for 10 min. We nicked the resulting circular pseudogene (1 ng/μl) by Nb.BsrDI and Nt.BspQI (0.5 U/μl, New England Biolabs) in 1× NEB3 buffer at 65°C for 2 h and we stopped the reaction by heating at 80°C for 20 min. We amplified the nicked circular pseudogene (0.1 ng/μl) at 30°C by RCA and T4 gene 32 (25 ng/μl; NEB) stopping the reaction by heat inactivation (80°C, for 20 min) at different time points.

    Techniques: Agarose Gel Electrophoresis, Amplification