t4 dna polymerase  (New England Biolabs)


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    Structured Review

    New England Biolabs t4 dna polymerase
    Nucleotide sequences of integrated oligonucleotide fragments. Sequences of integrated oligonucleotide fragments with features common to all LIC-LC1 and LIC-LC2 vectors are shown. Double-stranded oligonucleotides were integrated at the restriction enzyme recognition sites indicated except for PmeI which is used to eliminate the 670-bp stuffer fragment prior to the LIC process. LIC-pPICZ-LC1/-LC2 vectors were generated by inserting AclI/SalI-restricted double-stranded oligonucleotides into BstBI/SalI-digested expression vector (cutting with AclI and BstBI creates compatible 5′ overhangs), resulting in a change of the BstBI sequence (TTCGAA to TTCGTT). The asterisk on the forward strand indicates the position of adenine (corresponding to thymine on the reverse strand) required for the generation of LIC 5′ overhangs in the presence of <t>T4</t> DNA polymerase and dATP. The blue arrow indicates the TEV cleavage site suitable for the removal of the marker proteins IFP and 6xHis-tag.
    T4 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 216 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna polymerase/product/New England Biolabs
    Average 99 stars, based on 216 article reviews
    Price from $9.99 to $1999.99
    t4 dna polymerase - by Bioz Stars, 2020-01
    99/100 stars

    Images

    1) Product Images from "High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection"

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018900

    Nucleotide sequences of integrated oligonucleotide fragments. Sequences of integrated oligonucleotide fragments with features common to all LIC-LC1 and LIC-LC2 vectors are shown. Double-stranded oligonucleotides were integrated at the restriction enzyme recognition sites indicated except for PmeI which is used to eliminate the 670-bp stuffer fragment prior to the LIC process. LIC-pPICZ-LC1/-LC2 vectors were generated by inserting AclI/SalI-restricted double-stranded oligonucleotides into BstBI/SalI-digested expression vector (cutting with AclI and BstBI creates compatible 5′ overhangs), resulting in a change of the BstBI sequence (TTCGAA to TTCGTT). The asterisk on the forward strand indicates the position of adenine (corresponding to thymine on the reverse strand) required for the generation of LIC 5′ overhangs in the presence of T4 DNA polymerase and dATP. The blue arrow indicates the TEV cleavage site suitable for the removal of the marker proteins IFP and 6xHis-tag.
    Figure Legend Snippet: Nucleotide sequences of integrated oligonucleotide fragments. Sequences of integrated oligonucleotide fragments with features common to all LIC-LC1 and LIC-LC2 vectors are shown. Double-stranded oligonucleotides were integrated at the restriction enzyme recognition sites indicated except for PmeI which is used to eliminate the 670-bp stuffer fragment prior to the LIC process. LIC-pPICZ-LC1/-LC2 vectors were generated by inserting AclI/SalI-restricted double-stranded oligonucleotides into BstBI/SalI-digested expression vector (cutting with AclI and BstBI creates compatible 5′ overhangs), resulting in a change of the BstBI sequence (TTCGAA to TTCGTT). The asterisk on the forward strand indicates the position of adenine (corresponding to thymine on the reverse strand) required for the generation of LIC 5′ overhangs in the presence of T4 DNA polymerase and dATP. The blue arrow indicates the TEV cleavage site suitable for the removal of the marker proteins IFP and 6xHis-tag.

    Techniques Used: Generated, Expressing, Plasmid Preparation, Sequencing, Marker

    Ligation-independent cloning using LIC-IFP-compatible expression vectors. LIC vectors (LIC-LC1 and LIC-LC2) are cleaved with PmeI restriction enzyme and the released stuffer fragment (670 bp) is removed. The cleaved vector is treated with T4 DNA polymerase in the presence of dATP, whereas the PCR product (amplified open reading frame) is treated in the presence of dTTP. The asterisks indicate the position of adenine (vector) or thymine (PCR product) required for the generation of LIC-complementary 5′ overhangs. After successful annealing and transformation into E. coli , host-internal ligases and DNA polymerases close the vector and fill in the gaps, caused by the two additional nucleotides (CC, coloured in blue) upstream of the start codon (ATG), which are required to retain the reading frame. For LIC with LC1 vectors, PCR-amplified open reading frames contain a double stop codon (TAATAG); for LIC with LC2 vectors, open reading frames must not contain a stop codon to allow expression of ProteinX-TEV-IFP-6xHis fusion proteins. To provide the thymine moiety on the forward strand for dTTP/T4 DNA polymerase treatment, additional three nucleotides (GGT) are added directly at the 3′-end of the PCR-amplified open reading frame.
    Figure Legend Snippet: Ligation-independent cloning using LIC-IFP-compatible expression vectors. LIC vectors (LIC-LC1 and LIC-LC2) are cleaved with PmeI restriction enzyme and the released stuffer fragment (670 bp) is removed. The cleaved vector is treated with T4 DNA polymerase in the presence of dATP, whereas the PCR product (amplified open reading frame) is treated in the presence of dTTP. The asterisks indicate the position of adenine (vector) or thymine (PCR product) required for the generation of LIC-complementary 5′ overhangs. After successful annealing and transformation into E. coli , host-internal ligases and DNA polymerases close the vector and fill in the gaps, caused by the two additional nucleotides (CC, coloured in blue) upstream of the start codon (ATG), which are required to retain the reading frame. For LIC with LC1 vectors, PCR-amplified open reading frames contain a double stop codon (TAATAG); for LIC with LC2 vectors, open reading frames must not contain a stop codon to allow expression of ProteinX-TEV-IFP-6xHis fusion proteins. To provide the thymine moiety on the forward strand for dTTP/T4 DNA polymerase treatment, additional three nucleotides (GGT) are added directly at the 3′-end of the PCR-amplified open reading frame.

    Techniques Used: Ligation, Clone Assay, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Transformation Assay

    2) Product Images from "High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection"

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018900

    Nucleotide sequences of integrated oligonucleotide fragments. Sequences of integrated oligonucleotide fragments with features common to all LIC-LC1 and LIC-LC2 vectors are shown. Double-stranded oligonucleotides were integrated at the restriction enzyme recognition sites indicated except for PmeI which is used to eliminate the 670-bp stuffer fragment prior to the LIC process. LIC-pPICZ-LC1/-LC2 vectors were generated by inserting AclI/SalI-restricted double-stranded oligonucleotides into BstBI/SalI-digested expression vector (cutting with AclI and BstBI creates compatible 5′ overhangs), resulting in a change of the BstBI sequence (TTCGAA to TTCGTT). The asterisk on the forward strand indicates the position of adenine (corresponding to thymine on the reverse strand) required for the generation of LIC 5′ overhangs in the presence of T4 DNA polymerase and dATP. The blue arrow indicates the TEV cleavage site suitable for the removal of the marker proteins IFP and 6xHis-tag.
    Figure Legend Snippet: Nucleotide sequences of integrated oligonucleotide fragments. Sequences of integrated oligonucleotide fragments with features common to all LIC-LC1 and LIC-LC2 vectors are shown. Double-stranded oligonucleotides were integrated at the restriction enzyme recognition sites indicated except for PmeI which is used to eliminate the 670-bp stuffer fragment prior to the LIC process. LIC-pPICZ-LC1/-LC2 vectors were generated by inserting AclI/SalI-restricted double-stranded oligonucleotides into BstBI/SalI-digested expression vector (cutting with AclI and BstBI creates compatible 5′ overhangs), resulting in a change of the BstBI sequence (TTCGAA to TTCGTT). The asterisk on the forward strand indicates the position of adenine (corresponding to thymine on the reverse strand) required for the generation of LIC 5′ overhangs in the presence of T4 DNA polymerase and dATP. The blue arrow indicates the TEV cleavage site suitable for the removal of the marker proteins IFP and 6xHis-tag.

    Techniques Used: Generated, Expressing, Plasmid Preparation, Sequencing, Marker

    Ligation-independent cloning using LIC-IFP-compatible expression vectors. LIC vectors (LIC-LC1 and LIC-LC2) are cleaved with PmeI restriction enzyme and the released stuffer fragment (670 bp) is removed. The cleaved vector is treated with T4 DNA polymerase in the presence of dATP, whereas the PCR product (amplified open reading frame) is treated in the presence of dTTP. The asterisks indicate the position of adenine (vector) or thymine (PCR product) required for the generation of LIC-complementary 5′ overhangs. After successful annealing and transformation into E. coli , host-internal ligases and DNA polymerases close the vector and fill in the gaps, caused by the two additional nucleotides (CC, coloured in blue) upstream of the start codon (ATG), which are required to retain the reading frame. For LIC with LC1 vectors, PCR-amplified open reading frames contain a double stop codon (TAATAG); for LIC with LC2 vectors, open reading frames must not contain a stop codon to allow expression of ProteinX-TEV-IFP-6xHis fusion proteins. To provide the thymine moiety on the forward strand for dTTP/T4 DNA polymerase treatment, additional three nucleotides (GGT) are added directly at the 3′-end of the PCR-amplified open reading frame.
    Figure Legend Snippet: Ligation-independent cloning using LIC-IFP-compatible expression vectors. LIC vectors (LIC-LC1 and LIC-LC2) are cleaved with PmeI restriction enzyme and the released stuffer fragment (670 bp) is removed. The cleaved vector is treated with T4 DNA polymerase in the presence of dATP, whereas the PCR product (amplified open reading frame) is treated in the presence of dTTP. The asterisks indicate the position of adenine (vector) or thymine (PCR product) required for the generation of LIC-complementary 5′ overhangs. After successful annealing and transformation into E. coli , host-internal ligases and DNA polymerases close the vector and fill in the gaps, caused by the two additional nucleotides (CC, coloured in blue) upstream of the start codon (ATG), which are required to retain the reading frame. For LIC with LC1 vectors, PCR-amplified open reading frames contain a double stop codon (TAATAG); for LIC with LC2 vectors, open reading frames must not contain a stop codon to allow expression of ProteinX-TEV-IFP-6xHis fusion proteins. To provide the thymine moiety on the forward strand for dTTP/T4 DNA polymerase treatment, additional three nucleotides (GGT) are added directly at the 3′-end of the PCR-amplified open reading frame.

    Techniques Used: Ligation, Clone Assay, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Transformation Assay

    Related Articles

    Clone Assay:

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection
    Article Snippet: Paragraph title: Linearization of LIC expression vectors for LIC cloning ... To generate 5′ LIC overhangs (15 and 16 nt, respectively) at both ends the purified vector backbone was treated for 30 min (22°C) with T4 DNA polymerase in the presence of dATP, using the following reaction setup: 0.2 pmol purified vector backbone, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL dithiothreitol (DTT, 100 mM), 2 µL 10× (10 mg/mL) bovine serum albumin (BSA; NEB), 10 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O).

    Article Title: Re-evaluating the functional landscape of the cardiovascular system during development
    Article Snippet: The cloning-free method was also used to generate templates for gRNA synthesis ( ). .. The single strand DNA overhangs were filled with T4 DNA polymerase by adding 2.5 µl 10 mM dNTPs mix, 1 µl 10× NEBuffer2, 0.2 µl 100× NEB BSA and 0.5 µl T4 DNA polymerase (NEB) and then incubated at 12°C for 20 min.

    Amplification:

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection
    Article Snippet: Open reading frames of two cell wall degrading enzymes endo-β-1,4-glucanase (GenBank ID DQ490472) and endo-β-1,4-xylanase (DQ490490) were PCR amplified using genomic DNA from Pichia pastoris strains obtained from the Fungal Genetic Stock Centre (FGSC) . .. PCR products were treated at 22°C for 30 min with T4 DNA polymerase in the presence of dTTP, using the following reaction setup: 0.2 pmol purified PCR product, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL DTT (100 mM), 2 µL 10× BSA (10 mg/mL; NEB), 1 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O).

    In Vitro:

    Article Title: Re-evaluating the functional landscape of the cardiovascular system during development
    Article Snippet: The plasmid with gRNA target sequence was linearized by BamHI and used as a template of in vitro transcription reaction. gRNA was transcribed using MEGAshortscript kit (Ambion). .. The single strand DNA overhangs were filled with T4 DNA polymerase by adding 2.5 µl 10 mM dNTPs mix, 1 µl 10× NEBuffer2, 0.2 µl 100× NEB BSA and 0.5 µl T4 DNA polymerase (NEB) and then incubated at 12°C for 20 min.

    Mutagenesis:

    Article Title: Re-evaluating the functional landscape of the cardiovascular system during development
    Article Snippet: Paragraph title: CRISPR/CAS9 mutagenesis ... The single strand DNA overhangs were filled with T4 DNA polymerase by adding 2.5 µl 10 mM dNTPs mix, 1 µl 10× NEBuffer2, 0.2 µl 100× NEB BSA and 0.5 µl T4 DNA polymerase (NEB) and then incubated at 12°C for 20 min.

    Purification:

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection
    Article Snippet: .. To generate 5′ LIC overhangs (15 and 16 nt, respectively) at both ends the purified vector backbone was treated for 30 min (22°C) with T4 DNA polymerase in the presence of dATP, using the following reaction setup: 0.2 pmol purified vector backbone, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL dithiothreitol (DTT, 100 mM), 2 µL 10× (10 mg/mL) bovine serum albumin (BSA; NEB), 10 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O). .. The reaction mix was heat inactivated for 20 min at 75°C, followed by purification using the NucleoSpin Extract II kit (Macherey & Nagel) and elution with 20 µL elution buffer included in the kit.

    Article Title: Re-evaluating the functional landscape of the cardiovascular system during development
    Article Snippet: The single strand DNA overhangs were filled with T4 DNA polymerase by adding 2.5 µl 10 mM dNTPs mix, 1 µl 10× NEBuffer2, 0.2 µl 100× NEB BSA and 0.5 µl T4 DNA polymerase (NEB) and then incubated at 12°C for 20 min. .. The resulting double strand DNA was purified using QIAquick PCR purification kit (Qiagen).

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection
    Article Snippet: .. PCR products were treated at 22°C for 30 min with T4 DNA polymerase in the presence of dTTP, using the following reaction setup: 0.2 pmol purified PCR product, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL DTT (100 mM), 2 µL 10× BSA (10 mg/mL; NEB), 1 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O). .. The reaction mix was heat inactivated for 20 min at 75°C, followed by purification using the NucleoSpin Extract II kit (Macherey & Nagel) and eluting with 20 µL elution buffer included in the kit.

    Sequencing:

    Article Title: Re-evaluating the functional landscape of the cardiovascular system during development
    Article Snippet: The 1 µl of 100 µM gene-specific oligonucleotides containing T7 or SP6 sequence, 20 base target sequence without PAM, and a complementary region to constant oligonucleotide were mixed with 1 µl of 100 µM oligonucleotide encoding the reverse-complement of the tracrRNA tail with 1× NEBuffer2 in a total volume of 10 µl to anneal by the following procedure: denaturation for 5 min at 95°C, cooling to 85°C at −2°C/s and then cooling from 85°C to 25°C at −0.1°C/s. .. The single strand DNA overhangs were filled with T4 DNA polymerase by adding 2.5 µl 10 mM dNTPs mix, 1 µl 10× NEBuffer2, 0.2 µl 100× NEB BSA and 0.5 µl T4 DNA polymerase (NEB) and then incubated at 12°C for 20 min.

    Incubation:

    Article Title: Re-evaluating the functional landscape of the cardiovascular system during development
    Article Snippet: .. The single strand DNA overhangs were filled with T4 DNA polymerase by adding 2.5 µl 10 mM dNTPs mix, 1 µl 10× NEBuffer2, 0.2 µl 100× NEB BSA and 0.5 µl T4 DNA polymerase (NEB) and then incubated at 12°C for 20 min. .. The resulting double strand DNA was purified using QIAquick PCR purification kit (Qiagen).

    Expressing:

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection
    Article Snippet: Paragraph title: Linearization of LIC expression vectors for LIC cloning ... To generate 5′ LIC overhangs (15 and 16 nt, respectively) at both ends the purified vector backbone was treated for 30 min (22°C) with T4 DNA polymerase in the presence of dATP, using the following reaction setup: 0.2 pmol purified vector backbone, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL dithiothreitol (DTT, 100 mM), 2 µL 10× (10 mg/mL) bovine serum albumin (BSA; NEB), 10 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O).

    CRISPR:

    Article Title: Re-evaluating the functional landscape of the cardiovascular system during development
    Article Snippet: Paragraph title: CRISPR/CAS9 mutagenesis ... The single strand DNA overhangs were filled with T4 DNA polymerase by adding 2.5 µl 10 mM dNTPs mix, 1 µl 10× NEBuffer2, 0.2 µl 100× NEB BSA and 0.5 µl T4 DNA polymerase (NEB) and then incubated at 12°C for 20 min.

    Polymerase Chain Reaction:

    Article Title: Re-evaluating the functional landscape of the cardiovascular system during development
    Article Snippet: The single strand DNA overhangs were filled with T4 DNA polymerase by adding 2.5 µl 10 mM dNTPs mix, 1 µl 10× NEBuffer2, 0.2 µl 100× NEB BSA and 0.5 µl T4 DNA polymerase (NEB) and then incubated at 12°C for 20 min. .. The resulting double strand DNA was purified using QIAquick PCR purification kit (Qiagen).

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection
    Article Snippet: .. PCR products were treated at 22°C for 30 min with T4 DNA polymerase in the presence of dTTP, using the following reaction setup: 0.2 pmol purified PCR product, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL DTT (100 mM), 2 µL 10× BSA (10 mg/mL; NEB), 1 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O). .. The reaction mix was heat inactivated for 20 min at 75°C, followed by purification using the NucleoSpin Extract II kit (Macherey & Nagel) and eluting with 20 µL elution buffer included in the kit.

    Gel Extraction:

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection
    Article Snippet: Linearization of LIC expression vectors for LIC cloning LIC expression vectors (10 µg) were cut with 10 U PmeI in a 20-µL reaction volume and purified from contaminating stuffer fragment and undigested vector by gel-extraction using the NucleoSpin Extract II kit (Macherey & Nagel, Düren, Germany). .. To generate 5′ LIC overhangs (15 and 16 nt, respectively) at both ends the purified vector backbone was treated for 30 min (22°C) with T4 DNA polymerase in the presence of dATP, using the following reaction setup: 0.2 pmol purified vector backbone, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL dithiothreitol (DTT, 100 mM), 2 µL 10× (10 mg/mL) bovine serum albumin (BSA; NEB), 10 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O).

    Plasmid Preparation:

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection
    Article Snippet: .. To generate 5′ LIC overhangs (15 and 16 nt, respectively) at both ends the purified vector backbone was treated for 30 min (22°C) with T4 DNA polymerase in the presence of dATP, using the following reaction setup: 0.2 pmol purified vector backbone, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL dithiothreitol (DTT, 100 mM), 2 µL 10× (10 mg/mL) bovine serum albumin (BSA; NEB), 10 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O). .. The reaction mix was heat inactivated for 20 min at 75°C, followed by purification using the NucleoSpin Extract II kit (Macherey & Nagel) and elution with 20 µL elution buffer included in the kit.

    Article Title: Re-evaluating the functional landscape of the cardiovascular system during development
    Article Snippet: The plasmid with gRNA target sequence was linearized by BamHI and used as a template of in vitro transcription reaction. gRNA was transcribed using MEGAshortscript kit (Ambion). .. The single strand DNA overhangs were filled with T4 DNA polymerase by adding 2.5 µl 10 mM dNTPs mix, 1 µl 10× NEBuffer2, 0.2 µl 100× NEB BSA and 0.5 µl T4 DNA polymerase (NEB) and then incubated at 12°C for 20 min.

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    New England Biolabs dnase i
    Dnase I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 374 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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)
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    Image Search Results


    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

    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