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  • 99
    Zymo Research ez dna methylation kit
    Ez Dna Methylation Kit, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 20029 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ez dna methylation kit/product/Zymo Research
    Average 99 stars, based on 20029 article reviews
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    ez dna methylation kit - by Bioz Stars, 2021-01
    99/100 stars
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    99
    New England Biolabs t4 dna ligase
    T4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 49148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase/product/New England Biolabs
    Average 99 stars, based on 49148 article reviews
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    t4 dna ligase - by Bioz Stars, 2021-01
    99/100 stars
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    99
    Thermo Fisher taq polymerase
    Taq Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 33062 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq polymerase/product/Thermo Fisher
    Average 99 stars, based on 33062 article reviews
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    taq polymerase - by Bioz Stars, 2021-01
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    99
    Qiagen qiaamp dna mini kit
    Qiaamp Dna Mini Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 48891 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/qiaamp dna mini kit/product/Qiagen
    Average 99 stars, based on 48891 article reviews
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    qiaamp dna mini kit - by Bioz Stars, 2021-01
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    95
    Thermo Fisher 3730xl dna analyzer
    3730xl Dna Analyzer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 46651 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/3730xl dna analyzer/product/Thermo Fisher
    Average 95 stars, based on 46651 article reviews
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    3730xl dna analyzer - by Bioz Stars, 2021-01
    95/100 stars
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    99
    Thermo Fisher genomic dna
    Genomic Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 48727 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/genomic dna/product/Thermo Fisher
    Average 99 stars, based on 48727 article reviews
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    genomic dna - by Bioz Stars, 2021-01
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    99
    Thermo Fisher turbo dna free kit
    Turbo Dna Free Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 27396 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    turbo dna free kit - by Bioz Stars, 2021-01
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    99
    Qiagen qiaamp dna blood mini kit
    Qiaamp Dna Blood Mini Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 22255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 22255 article reviews
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    qiaamp dna blood mini kit - by Bioz Stars, 2021-01
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    99
    Thermo Fisher platinum taq polymerase
    Platinum Taq Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8048 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/platinum taq polymerase/product/Thermo Fisher
    Average 99 stars, based on 8048 article reviews
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    platinum taq polymerase - by Bioz Stars, 2021-01
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    99
    Thermo Fisher cdna synthesis
    Cdna Synthesis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 106131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cdna synthesis/product/Thermo Fisher
    Average 99 stars, based on 106131 article reviews
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    92
    Double Helix dna double helix
    (A) Diagram and sequence of CDKN2A region targeted by additional gRNAs. Due to the display of the reverse complement sequence of Fig. 3 , CpGs are numbered differently (black) but Fig. 3 numbering system is shown below in red. gRNA sequences are shown in green, where the entire green sequence represents the S. pyogenes gRNA and the addition 5’ nucleotide in black represents the additional nucleotide needed for S. aureus gRNAs. S. aureus PAM site is shown in blue, with the first 3 nucleotides (5’ to 3’) represent the NGG PAM of the S. pyogenes gRNA. (B) Each horizontal row depicts a heatmap of average <t>DNA</t> methylation at each numbered <t>CpG</t> over 10-20 (except SP-gRNA4, 4 clones) individual strands of DNA (bisulfite-converted clones) where light blue represents 0% methylation and dark red represents 100% methylation. The CpGs within the binding site of the labeled gRNA are labeled and enclosed in dashed lines. gRNAs1-3 interrogate DNA methylation interference of 5’ proximal CpGs and gRNA4 interrogates that of 3’ proximal CpGs. Lowly methylated strands of DNA (poor M.SssI methylation) and strands with unaffected binding sites (unbound by dCas9) were excluded from the analysis because efficacy was not under evaluation. (C-D) Data from (B) transformed into a percent methylation as a function of CpG distance in base pairs from the 5’ (C) or 3’ (D) end of the gRNA sequence (including PAM) and S. aureus (grey) or S. pyogenes (pink) across gRNAs 1-3 (C) or gRNA4 (D).
    Dna Double Helix, supplied by Double Helix, used in various techniques. Bioz Stars score: 92/100, based on 10708 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna double helix/product/Double Helix
    Average 92 stars, based on 10708 article reviews
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    dna double helix - by Bioz Stars, 2021-01
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    99
    Thermo Fisher t4 dna ligase
    (A) Diagram and sequence of CDKN2A region targeted by additional gRNAs. Due to the display of the reverse complement sequence of Fig. 3 , CpGs are numbered differently (black) but Fig. 3 numbering system is shown below in red. gRNA sequences are shown in green, where the entire green sequence represents the S. pyogenes gRNA and the addition 5’ nucleotide in black represents the additional nucleotide needed for S. aureus gRNAs. S. aureus PAM site is shown in blue, with the first 3 nucleotides (5’ to 3’) represent the NGG PAM of the S. pyogenes gRNA. (B) Each horizontal row depicts a heatmap of average <t>DNA</t> methylation at each numbered <t>CpG</t> over 10-20 (except SP-gRNA4, 4 clones) individual strands of DNA (bisulfite-converted clones) where light blue represents 0% methylation and dark red represents 100% methylation. The CpGs within the binding site of the labeled gRNA are labeled and enclosed in dashed lines. gRNAs1-3 interrogate DNA methylation interference of 5’ proximal CpGs and gRNA4 interrogates that of 3’ proximal CpGs. Lowly methylated strands of DNA (poor M.SssI methylation) and strands with unaffected binding sites (unbound by dCas9) were excluded from the analysis because efficacy was not under evaluation. (C-D) Data from (B) transformed into a percent methylation as a function of CpG distance in base pairs from the 5’ (C) or 3’ (D) end of the gRNA sequence (including PAM) and S. aureus (grey) or S. pyogenes (pink) across gRNAs 1-3 (C) or gRNA4 (D).
    T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25057 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase/product/Thermo Fisher
    Average 99 stars, based on 25057 article reviews
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    t4 dna ligase - by Bioz Stars, 2021-01
    99/100 stars
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    99
    New England Biolabs phusion high fidelity dna polymerase
    (A) Diagram and sequence of CDKN2A region targeted by additional gRNAs. Due to the display of the reverse complement sequence of Fig. 3 , CpGs are numbered differently (black) but Fig. 3 numbering system is shown below in red. gRNA sequences are shown in green, where the entire green sequence represents the S. pyogenes gRNA and the addition 5’ nucleotide in black represents the additional nucleotide needed for S. aureus gRNAs. S. aureus PAM site is shown in blue, with the first 3 nucleotides (5’ to 3’) represent the NGG PAM of the S. pyogenes gRNA. (B) Each horizontal row depicts a heatmap of average <t>DNA</t> methylation at each numbered <t>CpG</t> over 10-20 (except SP-gRNA4, 4 clones) individual strands of DNA (bisulfite-converted clones) where light blue represents 0% methylation and dark red represents 100% methylation. The CpGs within the binding site of the labeled gRNA are labeled and enclosed in dashed lines. gRNAs1-3 interrogate DNA methylation interference of 5’ proximal CpGs and gRNA4 interrogates that of 3’ proximal CpGs. Lowly methylated strands of DNA (poor M.SssI methylation) and strands with unaffected binding sites (unbound by dCas9) were excluded from the analysis because efficacy was not under evaluation. (C-D) Data from (B) transformed into a percent methylation as a function of CpG distance in base pairs from the 5’ (C) or 3’ (D) end of the gRNA sequence (including PAM) and S. aureus (grey) or S. pyogenes (pink) across gRNAs 1-3 (C) or gRNA4 (D).
    Phusion High Fidelity Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 23530 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phusion high fidelity dna polymerase/product/New England Biolabs
    Average 99 stars, based on 23530 article reviews
    Price from $9.99 to $1999.99
    phusion high fidelity dna polymerase - by Bioz Stars, 2021-01
    99/100 stars
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    99
    TaKaRa taq polymerase
    (A) Diagram and sequence of CDKN2A region targeted by additional gRNAs. Due to the display of the reverse complement sequence of Fig. 3 , CpGs are numbered differently (black) but Fig. 3 numbering system is shown below in red. gRNA sequences are shown in green, where the entire green sequence represents the S. pyogenes gRNA and the addition 5’ nucleotide in black represents the additional nucleotide needed for S. aureus gRNAs. S. aureus PAM site is shown in blue, with the first 3 nucleotides (5’ to 3’) represent the NGG PAM of the S. pyogenes gRNA. (B) Each horizontal row depicts a heatmap of average <t>DNA</t> methylation at each numbered <t>CpG</t> over 10-20 (except SP-gRNA4, 4 clones) individual strands of DNA (bisulfite-converted clones) where light blue represents 0% methylation and dark red represents 100% methylation. The CpGs within the binding site of the labeled gRNA are labeled and enclosed in dashed lines. gRNAs1-3 interrogate DNA methylation interference of 5’ proximal CpGs and gRNA4 interrogates that of 3’ proximal CpGs. Lowly methylated strands of DNA (poor M.SssI methylation) and strands with unaffected binding sites (unbound by dCas9) were excluded from the analysis because efficacy was not under evaluation. (C-D) Data from (B) transformed into a percent methylation as a function of CpG distance in base pairs from the 5’ (C) or 3’ (D) end of the gRNA sequence (including PAM) and S. aureus (grey) or S. pyogenes (pink) across gRNAs 1-3 (C) or gRNA4 (D).
    Taq Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 10064 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq polymerase/product/TaKaRa
    Average 99 stars, based on 10064 article reviews
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    taq polymerase - by Bioz Stars, 2021-01
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    99
    New England Biolabs taq polymerase
    (A) Diagram and sequence of CDKN2A region targeted by additional gRNAs. Due to the display of the reverse complement sequence of Fig. 3 , CpGs are numbered differently (black) but Fig. 3 numbering system is shown below in red. gRNA sequences are shown in green, where the entire green sequence represents the S. pyogenes gRNA and the addition 5’ nucleotide in black represents the additional nucleotide needed for S. aureus gRNAs. S. aureus PAM site is shown in blue, with the first 3 nucleotides (5’ to 3’) represent the NGG PAM of the S. pyogenes gRNA. (B) Each horizontal row depicts a heatmap of average <t>DNA</t> methylation at each numbered <t>CpG</t> over 10-20 (except SP-gRNA4, 4 clones) individual strands of DNA (bisulfite-converted clones) where light blue represents 0% methylation and dark red represents 100% methylation. The CpGs within the binding site of the labeled gRNA are labeled and enclosed in dashed lines. gRNAs1-3 interrogate DNA methylation interference of 5’ proximal CpGs and gRNA4 interrogates that of 3’ proximal CpGs. Lowly methylated strands of DNA (poor M.SssI methylation) and strands with unaffected binding sites (unbound by dCas9) were excluded from the analysis because efficacy was not under evaluation. (C-D) Data from (B) transformed into a percent methylation as a function of CpG distance in base pairs from the 5’ (C) or 3’ (D) end of the gRNA sequence (including PAM) and S. aureus (grey) or S. pyogenes (pink) across gRNAs 1-3 (C) or gRNA4 (D).
    Taq Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 8780 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq polymerase/product/New England Biolabs
    Average 99 stars, based on 8780 article reviews
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    taq polymerase - by Bioz Stars, 2021-01
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    99
    Thermo Fisher phusion high fidelity dna polymerase
    (A) Diagram and sequence of CDKN2A region targeted by additional gRNAs. Due to the display of the reverse complement sequence of Fig. 3 , CpGs are numbered differently (black) but Fig. 3 numbering system is shown below in red. gRNA sequences are shown in green, where the entire green sequence represents the S. pyogenes gRNA and the addition 5’ nucleotide in black represents the additional nucleotide needed for S. aureus gRNAs. S. aureus PAM site is shown in blue, with the first 3 nucleotides (5’ to 3’) represent the NGG PAM of the S. pyogenes gRNA. (B) Each horizontal row depicts a heatmap of average <t>DNA</t> methylation at each numbered <t>CpG</t> over 10-20 (except SP-gRNA4, 4 clones) individual strands of DNA (bisulfite-converted clones) where light blue represents 0% methylation and dark red represents 100% methylation. The CpGs within the binding site of the labeled gRNA are labeled and enclosed in dashed lines. gRNAs1-3 interrogate DNA methylation interference of 5’ proximal CpGs and gRNA4 interrogates that of 3’ proximal CpGs. Lowly methylated strands of DNA (poor M.SssI methylation) and strands with unaffected binding sites (unbound by dCas9) were excluded from the analysis because efficacy was not under evaluation. (C-D) Data from (B) transformed into a percent methylation as a function of CpG distance in base pairs from the 5’ (C) or 3’ (D) end of the gRNA sequence (including PAM) and S. aureus (grey) or S. pyogenes (pink) across gRNAs 1-3 (C) or gRNA4 (D).
    Phusion High Fidelity Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18947 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phusion high fidelity dna polymerase/product/Thermo Fisher
    Average 99 stars, based on 18947 article reviews
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    phusion high fidelity dna polymerase - by Bioz Stars, 2021-01
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    99
    Zymo Research ez dna methylation gold kit
    (A) Diagram and sequence of CDKN2A region targeted by additional gRNAs. Due to the display of the reverse complement sequence of Fig. 3 , CpGs are numbered differently (black) but Fig. 3 numbering system is shown below in red. gRNA sequences are shown in green, where the entire green sequence represents the S. pyogenes gRNA and the addition 5’ nucleotide in black represents the additional nucleotide needed for S. aureus gRNAs. S. aureus PAM site is shown in blue, with the first 3 nucleotides (5’ to 3’) represent the NGG PAM of the S. pyogenes gRNA. (B) Each horizontal row depicts a heatmap of average <t>DNA</t> methylation at each numbered <t>CpG</t> over 10-20 (except SP-gRNA4, 4 clones) individual strands of DNA (bisulfite-converted clones) where light blue represents 0% methylation and dark red represents 100% methylation. The CpGs within the binding site of the labeled gRNA are labeled and enclosed in dashed lines. gRNAs1-3 interrogate DNA methylation interference of 5’ proximal CpGs and gRNA4 interrogates that of 3’ proximal CpGs. Lowly methylated strands of DNA (poor M.SssI methylation) and strands with unaffected binding sites (unbound by dCas9) were excluded from the analysis because efficacy was not under evaluation. (C-D) Data from (B) transformed into a percent methylation as a function of CpG distance in base pairs from the 5’ (C) or 3’ (D) end of the gRNA sequence (including PAM) and S. aureus (grey) or S. pyogenes (pink) across gRNAs 1-3 (C) or gRNA4 (D).
    Ez Dna Methylation Gold Kit, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 16191 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ez dna methylation gold kit/product/Zymo Research
    Average 99 stars, based on 16191 article reviews
    Price from $9.99 to $1999.99
    ez dna methylation gold kit - by Bioz Stars, 2021-01
    99/100 stars
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    99
    TaKaRa ex taq dna polymerase
    (A) Diagram and sequence of CDKN2A region targeted by additional gRNAs. Due to the display of the reverse complement sequence of Fig. 3 , CpGs are numbered differently (black) but Fig. 3 numbering system is shown below in red. gRNA sequences are shown in green, where the entire green sequence represents the S. pyogenes gRNA and the addition 5’ nucleotide in black represents the additional nucleotide needed for S. aureus gRNAs. S. aureus PAM site is shown in blue, with the first 3 nucleotides (5’ to 3’) represent the NGG PAM of the S. pyogenes gRNA. (B) Each horizontal row depicts a heatmap of average <t>DNA</t> methylation at each numbered <t>CpG</t> over 10-20 (except SP-gRNA4, 4 clones) individual strands of DNA (bisulfite-converted clones) where light blue represents 0% methylation and dark red represents 100% methylation. The CpGs within the binding site of the labeled gRNA are labeled and enclosed in dashed lines. gRNAs1-3 interrogate DNA methylation interference of 5’ proximal CpGs and gRNA4 interrogates that of 3’ proximal CpGs. Lowly methylated strands of DNA (poor M.SssI methylation) and strands with unaffected binding sites (unbound by dCas9) were excluded from the analysis because efficacy was not under evaluation. (C-D) Data from (B) transformed into a percent methylation as a function of CpG distance in base pairs from the 5’ (C) or 3’ (D) end of the gRNA sequence (including PAM) and S. aureus (grey) or S. pyogenes (pink) across gRNAs 1-3 (C) or gRNA4 (D).
    Ex Taq Dna Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 9323 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ex taq dna polymerase/product/TaKaRa
    Average 99 stars, based on 9323 article reviews
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    ex taq dna polymerase - by Bioz Stars, 2021-01
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    99
    Qiagen qiaamp dna stool mini kit
    (A) Diagram and sequence of CDKN2A region targeted by additional gRNAs. Due to the display of the reverse complement sequence of Fig. 3 , CpGs are numbered differently (black) but Fig. 3 numbering system is shown below in red. gRNA sequences are shown in green, where the entire green sequence represents the S. pyogenes gRNA and the addition 5’ nucleotide in black represents the additional nucleotide needed for S. aureus gRNAs. S. aureus PAM site is shown in blue, with the first 3 nucleotides (5’ to 3’) represent the NGG PAM of the S. pyogenes gRNA. (B) Each horizontal row depicts a heatmap of average <t>DNA</t> methylation at each numbered <t>CpG</t> over 10-20 (except SP-gRNA4, 4 clones) individual strands of DNA (bisulfite-converted clones) where light blue represents 0% methylation and dark red represents 100% methylation. The CpGs within the binding site of the labeled gRNA are labeled and enclosed in dashed lines. gRNAs1-3 interrogate DNA methylation interference of 5’ proximal CpGs and gRNA4 interrogates that of 3’ proximal CpGs. Lowly methylated strands of DNA (poor M.SssI methylation) and strands with unaffected binding sites (unbound by dCas9) were excluded from the analysis because efficacy was not under evaluation. (C-D) Data from (B) transformed into a percent methylation as a function of CpG distance in base pairs from the 5’ (C) or 3’ (D) end of the gRNA sequence (including PAM) and S. aureus (grey) or S. pyogenes (pink) across gRNAs 1-3 (C) or gRNA4 (D).
    Qiaamp Dna Stool Mini Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 10372 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/qiaamp dna stool mini kit/product/Qiagen
    Average 99 stars, based on 10372 article reviews
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    qiaamp dna stool mini kit - by Bioz Stars, 2021-01
    99/100 stars
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    99
    Qiagen taq dna polymerase
    (A) Diagram and sequence of CDKN2A region targeted by additional gRNAs. Due to the display of the reverse complement sequence of Fig. 3 , CpGs are numbered differently (black) but Fig. 3 numbering system is shown below in red. gRNA sequences are shown in green, where the entire green sequence represents the S. pyogenes gRNA and the addition 5’ nucleotide in black represents the additional nucleotide needed for S. aureus gRNAs. S. aureus PAM site is shown in blue, with the first 3 nucleotides (5’ to 3’) represent the NGG PAM of the S. pyogenes gRNA. (B) Each horizontal row depicts a heatmap of average <t>DNA</t> methylation at each numbered <t>CpG</t> over 10-20 (except SP-gRNA4, 4 clones) individual strands of DNA (bisulfite-converted clones) where light blue represents 0% methylation and dark red represents 100% methylation. The CpGs within the binding site of the labeled gRNA are labeled and enclosed in dashed lines. gRNAs1-3 interrogate DNA methylation interference of 5’ proximal CpGs and gRNA4 interrogates that of 3’ proximal CpGs. Lowly methylated strands of DNA (poor M.SssI methylation) and strands with unaffected binding sites (unbound by dCas9) were excluded from the analysis because efficacy was not under evaluation. (C-D) Data from (B) transformed into a percent methylation as a function of CpG distance in base pairs from the 5’ (C) or 3’ (D) end of the gRNA sequence (including PAM) and S. aureus (grey) or S. pyogenes (pink) across gRNAs 1-3 (C) or gRNA4 (D).
    Taq Dna Polymerase, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 8578 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq dna polymerase/product/Qiagen
    Average 99 stars, based on 8578 article reviews
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    taq dna polymerase - by Bioz Stars, 2021-01
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    99
    Thermo Fisher dna free kit
    (A) Diagram and sequence of CDKN2A region targeted by additional gRNAs. Due to the display of the reverse complement sequence of Fig. 3 , CpGs are numbered differently (black) but Fig. 3 numbering system is shown below in red. gRNA sequences are shown in green, where the entire green sequence represents the S. pyogenes gRNA and the addition 5’ nucleotide in black represents the additional nucleotide needed for S. aureus gRNAs. S. aureus PAM site is shown in blue, with the first 3 nucleotides (5’ to 3’) represent the NGG PAM of the S. pyogenes gRNA. (B) Each horizontal row depicts a heatmap of average <t>DNA</t> methylation at each numbered <t>CpG</t> over 10-20 (except SP-gRNA4, 4 clones) individual strands of DNA (bisulfite-converted clones) where light blue represents 0% methylation and dark red represents 100% methylation. The CpGs within the binding site of the labeled gRNA are labeled and enclosed in dashed lines. gRNAs1-3 interrogate DNA methylation interference of 5’ proximal CpGs and gRNA4 interrogates that of 3’ proximal CpGs. Lowly methylated strands of DNA (poor M.SssI methylation) and strands with unaffected binding sites (unbound by dCas9) were excluded from the analysis because efficacy was not under evaluation. (C-D) Data from (B) transformed into a percent methylation as a function of CpG distance in base pairs from the 5’ (C) or 3’ (D) end of the gRNA sequence (including PAM) and S. aureus (grey) or S. pyogenes (pink) across gRNAs 1-3 (C) or gRNA4 (D).
    Dna Free Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 13017 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna free kit/product/Thermo Fisher
    Average 99 stars, based on 13017 article reviews
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    dna free kit - by Bioz Stars, 2021-01
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    99
    Thermo Fisher dna concentration
    (A) Diagram and sequence of CDKN2A region targeted by additional gRNAs. Due to the display of the reverse complement sequence of Fig. 3 , CpGs are numbered differently (black) but Fig. 3 numbering system is shown below in red. gRNA sequences are shown in green, where the entire green sequence represents the S. pyogenes gRNA and the addition 5’ nucleotide in black represents the additional nucleotide needed for S. aureus gRNAs. S. aureus PAM site is shown in blue, with the first 3 nucleotides (5’ to 3’) represent the NGG PAM of the S. pyogenes gRNA. (B) Each horizontal row depicts a heatmap of average <t>DNA</t> methylation at each numbered <t>CpG</t> over 10-20 (except SP-gRNA4, 4 clones) individual strands of DNA (bisulfite-converted clones) where light blue represents 0% methylation and dark red represents 100% methylation. The CpGs within the binding site of the labeled gRNA are labeled and enclosed in dashed lines. gRNAs1-3 interrogate DNA methylation interference of 5’ proximal CpGs and gRNA4 interrogates that of 3’ proximal CpGs. Lowly methylated strands of DNA (poor M.SssI methylation) and strands with unaffected binding sites (unbound by dCas9) were excluded from the analysis because efficacy was not under evaluation. (C-D) Data from (B) transformed into a percent methylation as a function of CpG distance in base pairs from the 5’ (C) or 3’ (D) end of the gRNA sequence (including PAM) and S. aureus (grey) or S. pyogenes (pink) across gRNAs 1-3 (C) or gRNA4 (D).
    Dna Concentration, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3549 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna concentration/product/Thermo Fisher
    Average 99 stars, based on 3549 article reviews
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    dna concentration - by Bioz Stars, 2021-01
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    99
    Qiagen qiaamp dna ffpe tissue kit
    Clinical observation of <t>DNA</t> quality index (DQI) in 211 consecutive surplus samples. The DNA quality of 211 consecutive surplus specimens-including 130 <t>FFPE</t> tissue blocks (FFPE Tissue), 27 FFPE cell blocks of FNA samples (FFPE FNA), 29 freshly centrifuged pleural fluid samples (fresh pleural fluid), and 25 FFPE cell blocks of pleural fluid (FFPE pleural fluid)-was evaluated. The DNA Quality Index and amplifiable DNA concentrations were determined using a DNA Quality Index Kit (Beijing ACCB Biotech Ltd., Beijing, China) according to the manufacturer’s instructions.
    Qiaamp Dna Ffpe Tissue Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 10487 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/qiaamp dna ffpe tissue kit/product/Qiagen
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    Clinical observation of <t>DNA</t> quality index (DQI) in 211 consecutive surplus samples. The DNA quality of 211 consecutive surplus specimens-including 130 <t>FFPE</t> tissue blocks (FFPE Tissue), 27 FFPE cell blocks of FNA samples (FFPE FNA), 29 freshly centrifuged pleural fluid samples (fresh pleural fluid), and 25 FFPE cell blocks of pleural fluid (FFPE pleural fluid)-was evaluated. The DNA Quality Index and amplifiable DNA concentrations were determined using a DNA Quality Index Kit (Beijing ACCB Biotech Ltd., Beijing, China) according to the manufacturer’s instructions.
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    Image Search Results


    (A) Diagram and sequence of CDKN2A region targeted by additional gRNAs. Due to the display of the reverse complement sequence of Fig. 3 , CpGs are numbered differently (black) but Fig. 3 numbering system is shown below in red. gRNA sequences are shown in green, where the entire green sequence represents the S. pyogenes gRNA and the addition 5’ nucleotide in black represents the additional nucleotide needed for S. aureus gRNAs. S. aureus PAM site is shown in blue, with the first 3 nucleotides (5’ to 3’) represent the NGG PAM of the S. pyogenes gRNA. (B) Each horizontal row depicts a heatmap of average DNA methylation at each numbered CpG over 10-20 (except SP-gRNA4, 4 clones) individual strands of DNA (bisulfite-converted clones) where light blue represents 0% methylation and dark red represents 100% methylation. The CpGs within the binding site of the labeled gRNA are labeled and enclosed in dashed lines. gRNAs1-3 interrogate DNA methylation interference of 5’ proximal CpGs and gRNA4 interrogates that of 3’ proximal CpGs. Lowly methylated strands of DNA (poor M.SssI methylation) and strands with unaffected binding sites (unbound by dCas9) were excluded from the analysis because efficacy was not under evaluation. (C-D) Data from (B) transformed into a percent methylation as a function of CpG distance in base pairs from the 5’ (C) or 3’ (D) end of the gRNA sequence (including PAM) and S. aureus (grey) or S. pyogenes (pink) across gRNAs 1-3 (C) or gRNA4 (D).

    Journal: bioRxiv

    Article Title: Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

    doi: 10.1101/2020.03.28.012518

    Figure Lengend Snippet: (A) Diagram and sequence of CDKN2A region targeted by additional gRNAs. Due to the display of the reverse complement sequence of Fig. 3 , CpGs are numbered differently (black) but Fig. 3 numbering system is shown below in red. gRNA sequences are shown in green, where the entire green sequence represents the S. pyogenes gRNA and the addition 5’ nucleotide in black represents the additional nucleotide needed for S. aureus gRNAs. S. aureus PAM site is shown in blue, with the first 3 nucleotides (5’ to 3’) represent the NGG PAM of the S. pyogenes gRNA. (B) Each horizontal row depicts a heatmap of average DNA methylation at each numbered CpG over 10-20 (except SP-gRNA4, 4 clones) individual strands of DNA (bisulfite-converted clones) where light blue represents 0% methylation and dark red represents 100% methylation. The CpGs within the binding site of the labeled gRNA are labeled and enclosed in dashed lines. gRNAs1-3 interrogate DNA methylation interference of 5’ proximal CpGs and gRNA4 interrogates that of 3’ proximal CpGs. Lowly methylated strands of DNA (poor M.SssI methylation) and strands with unaffected binding sites (unbound by dCas9) were excluded from the analysis because efficacy was not under evaluation. (C-D) Data from (B) transformed into a percent methylation as a function of CpG distance in base pairs from the 5’ (C) or 3’ (D) end of the gRNA sequence (including PAM) and S. aureus (grey) or S. pyogenes (pink) across gRNAs 1-3 (C) or gRNA4 (D).

    Article Snippet: Given that bound dCas9 envelopes nearly the entire DNA double helix [ ], we predicted that both CpG sites would be equally protected.

    Techniques: Sequencing, DNA Methylation Assay, Methylation, Binding Assay, Labeling, Transformation Assay

    (A) Bisulfite-cloning and sanger sequencing analysis of the Il33-002 promoter in NIH-3T3 cells treated with 1 µg/mL poly(I:C) or water control for 8 or 24 hours. Each horizontal row is one strand of DNA. Numbers indicate the CpG in the promoter. Red squares indicate methylated CpGs, blue squares indicate unmethylated CpGs, and white squares indicate a lack of data due to sequencing failure. (B) Il33-002 expression in 50nM TSA or vehicle (DMSO) treated NIH-3T3 cell lines stably expressing gRNAscr, gRNA1, gRNA2, or gRNA3 under high-puromycin conditions in combination with dCas9, followed by dCas9 removal by Cre recombinase as assayed by qRT-PCR and normalized to Actb expression (n=3). (C) Il33-002 expression in 100 ng/mL LPS or vehicle (PBS) treated NIH-3T3 cell lines stably expressing gRNAscr or gRNA3 under high-puromycin conditions in combination with dCas9, followed by dCas9 removal by Cre recombinase, as assayed by qRT-PCR and normalized to Actb expression, either 1 or 3 hours after treatment and displayed relative to expression measured at time=0 (n=3). * indicates statistically significant difference of p

    Journal: bioRxiv

    Article Title: Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

    doi: 10.1101/2020.03.28.012518

    Figure Lengend Snippet: (A) Bisulfite-cloning and sanger sequencing analysis of the Il33-002 promoter in NIH-3T3 cells treated with 1 µg/mL poly(I:C) or water control for 8 or 24 hours. Each horizontal row is one strand of DNA. Numbers indicate the CpG in the promoter. Red squares indicate methylated CpGs, blue squares indicate unmethylated CpGs, and white squares indicate a lack of data due to sequencing failure. (B) Il33-002 expression in 50nM TSA or vehicle (DMSO) treated NIH-3T3 cell lines stably expressing gRNAscr, gRNA1, gRNA2, or gRNA3 under high-puromycin conditions in combination with dCas9, followed by dCas9 removal by Cre recombinase as assayed by qRT-PCR and normalized to Actb expression (n=3). (C) Il33-002 expression in 100 ng/mL LPS or vehicle (PBS) treated NIH-3T3 cell lines stably expressing gRNAscr or gRNA3 under high-puromycin conditions in combination with dCas9, followed by dCas9 removal by Cre recombinase, as assayed by qRT-PCR and normalized to Actb expression, either 1 or 3 hours after treatment and displayed relative to expression measured at time=0 (n=3). * indicates statistically significant difference of p

    Article Snippet: Given that bound dCas9 envelopes nearly the entire DNA double helix [ ], we predicted that both CpG sites would be equally protected.

    Techniques: Clone Assay, Sequencing, Methylation, Expressing, Stable Transfection, Quantitative RT-PCR

    The footprint of dCas9 (A) Genome browser diagram of the CDKN2A (p16) promoter region, which was used for the methylation assay, showing transcription start site (TSS, marked by black arrow), gRNA position overlapping CpG 17, and surrounding CpGs. Below, DNA sequence is shown in black, gRNA sequence in blue, and PAM site in red, with CpGs bolded, underlined, and numbered according to the figures that follow. (B-E) Methylation of individual strands of the CDKN2A promoter plasmid following standard methylation (B,D) or methylation preceded by incubation with dCas9 and p16 gRNA (C,E). Red squares indicate methylated CpGs and blue squares indicate unmethylated CpGs; white squares indicate no data. Figures (B) and (C) represent the forward strand whereas (D) and (E) represent the reverse strand. Figures generated by BISMA software ( http://services.ibc.uni-stuttgart.de/BDPC/BISMA/ ). Regions below 80% methylation were filtered out as strands that were not effectively methylated by M.SssI.

    Journal: bioRxiv

    Article Title: Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

    doi: 10.1101/2020.03.28.012518

    Figure Lengend Snippet: The footprint of dCas9 (A) Genome browser diagram of the CDKN2A (p16) promoter region, which was used for the methylation assay, showing transcription start site (TSS, marked by black arrow), gRNA position overlapping CpG 17, and surrounding CpGs. Below, DNA sequence is shown in black, gRNA sequence in blue, and PAM site in red, with CpGs bolded, underlined, and numbered according to the figures that follow. (B-E) Methylation of individual strands of the CDKN2A promoter plasmid following standard methylation (B,D) or methylation preceded by incubation with dCas9 and p16 gRNA (C,E). Red squares indicate methylated CpGs and blue squares indicate unmethylated CpGs; white squares indicate no data. Figures (B) and (C) represent the forward strand whereas (D) and (E) represent the reverse strand. Figures generated by BISMA software ( http://services.ibc.uni-stuttgart.de/BDPC/BISMA/ ). Regions below 80% methylation were filtered out as strands that were not effectively methylated by M.SssI.

    Article Snippet: Given that bound dCas9 envelopes nearly the entire DNA double helix [ ], we predicted that both CpG sites would be equally protected.

    Techniques: Methylation, Sequencing, Plasmid Preparation, Incubation, Generated, Software

    (A) Genome browser view of the murine Tnf locus;(Top) each CG location marked by a blue dash and numbered below, TSS indicated by a black arrow., and the location of gRNA Tnf2 is labeled with a red line and marked accordingly; (Bottom) Two known distal enhancers of Tnf expression indicated with purple boxes, named and marked with distances to Tnf TSS [ 67 ]. (B) Table demonstrating the average methylation of Tnf CpGs numbered in (A) as measured by bisulfite-pyrosequencing a function of six candidate Tnf -targeting gRNAs or gRNAscr control in NIH-3T3 cells also stably expressing dCas9. CpGs within the gRNA binding site are indicated below the gRNA number and their methylation status is highlighted in yellow in the corresponding gRNAs. (C) DNA methylation levels assessed by bisulfite-pyrosequencing of NIH-3T3 cells stably expressing dCas9 and either gRNA Tnf2 (pink) or gRNAscr (grey) (n=3). (D) Tnf expression in NIH-3T3 cell lines subcloned from those in Fig. 6H stably expressing gRNAscr (grey) or gRNA Tnf2 (pink) under high-puromycin conditions in combination with dCas9, followed by dCas9 removal by Cre recombinase as assayed by RT-qPCR and normalized to Actb expression (n=8-9). * indicates statistically significant difference of p

    Journal: bioRxiv

    Article Title: Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

    doi: 10.1101/2020.03.28.012518

    Figure Lengend Snippet: (A) Genome browser view of the murine Tnf locus;(Top) each CG location marked by a blue dash and numbered below, TSS indicated by a black arrow., and the location of gRNA Tnf2 is labeled with a red line and marked accordingly; (Bottom) Two known distal enhancers of Tnf expression indicated with purple boxes, named and marked with distances to Tnf TSS [ 67 ]. (B) Table demonstrating the average methylation of Tnf CpGs numbered in (A) as measured by bisulfite-pyrosequencing a function of six candidate Tnf -targeting gRNAs or gRNAscr control in NIH-3T3 cells also stably expressing dCas9. CpGs within the gRNA binding site are indicated below the gRNA number and their methylation status is highlighted in yellow in the corresponding gRNAs. (C) DNA methylation levels assessed by bisulfite-pyrosequencing of NIH-3T3 cells stably expressing dCas9 and either gRNA Tnf2 (pink) or gRNAscr (grey) (n=3). (D) Tnf expression in NIH-3T3 cell lines subcloned from those in Fig. 6H stably expressing gRNAscr (grey) or gRNA Tnf2 (pink) under high-puromycin conditions in combination with dCas9, followed by dCas9 removal by Cre recombinase as assayed by RT-qPCR and normalized to Actb expression (n=8-9). * indicates statistically significant difference of p

    Article Snippet: Given that bound dCas9 envelopes nearly the entire DNA double helix [ ], we predicted that both CpG sites would be equally protected.

    Techniques: Labeling, Expressing, Methylation, Stable Transfection, Binding Assay, DNA Methylation Assay, Quantitative RT-PCR

    dCas9 causes demethylation in mammalian cells (A-C) Methylation levels assayed by bisulfite-pyrosequencing at CpGs 1, 2, 3, 5, 9, 10, and 11 of NIH-3T3 cells stably expressing dCas9 and gRNA1 (A, blue), gRNA2 (B, purple), gRNA3 (C, pink) or scrambled gRNA (A-C, grey; identical in all). Data is displayed as mean +/- SEM. (D). Cells from (C) were passaged for an additional 30 days and methylation percentage was assayed as previously (n=3, mean +/- SEM). (E) Cells from (C) were subjected to clonal isolation and expansion. Grey circles represent methylation levels of clones containing dCas9 and scrambled gRNA and various red circles represent methylation levels of randomly selected clones stably expressing dCas9 and gRNA 3 (n=10 per condition). (F) Average DNA methylation at CpGs 9-11, assayed by bisulfite-pyrosequencing, as a function of increasing the selection antibiotic puromycin (lentivirus is expressing puromycin resistance gene) concentration in cell lines (pools) stably expressing dCas9 and gRNA3 (n=1 per puromycin concentration) fitted with a line of best fit. (G). DNA methylation at CpGs 1, 2, 3, 9, 10, and 11 in NIH-3T3 cells stably expressing dCas9 and gRNA3 (pink) or control gRNAscr (grey) and treated with 10 µg/mL puromycin until no antibiotic-associated cell death could be observed and surviving cells were of sufficient quantity for DNA extraction and other procedures (approximately 2 weeks). * indicates statistically significant difference of p

    Journal: bioRxiv

    Article Title: Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

    doi: 10.1101/2020.03.28.012518

    Figure Lengend Snippet: dCas9 causes demethylation in mammalian cells (A-C) Methylation levels assayed by bisulfite-pyrosequencing at CpGs 1, 2, 3, 5, 9, 10, and 11 of NIH-3T3 cells stably expressing dCas9 and gRNA1 (A, blue), gRNA2 (B, purple), gRNA3 (C, pink) or scrambled gRNA (A-C, grey; identical in all). Data is displayed as mean +/- SEM. (D). Cells from (C) were passaged for an additional 30 days and methylation percentage was assayed as previously (n=3, mean +/- SEM). (E) Cells from (C) were subjected to clonal isolation and expansion. Grey circles represent methylation levels of clones containing dCas9 and scrambled gRNA and various red circles represent methylation levels of randomly selected clones stably expressing dCas9 and gRNA 3 (n=10 per condition). (F) Average DNA methylation at CpGs 9-11, assayed by bisulfite-pyrosequencing, as a function of increasing the selection antibiotic puromycin (lentivirus is expressing puromycin resistance gene) concentration in cell lines (pools) stably expressing dCas9 and gRNA3 (n=1 per puromycin concentration) fitted with a line of best fit. (G). DNA methylation at CpGs 1, 2, 3, 9, 10, and 11 in NIH-3T3 cells stably expressing dCas9 and gRNA3 (pink) or control gRNAscr (grey) and treated with 10 µg/mL puromycin until no antibiotic-associated cell death could be observed and surviving cells were of sufficient quantity for DNA extraction and other procedures (approximately 2 weeks). * indicates statistically significant difference of p

    Article Snippet: Given that bound dCas9 envelopes nearly the entire DNA double helix [ ], we predicted that both CpG sites would be equally protected.

    Techniques: Methylation, Stable Transfection, Expressing, Isolation, Clone Assay, DNA Methylation Assay, Selection, Concentration Assay, DNA Extraction

    Targeting the Il33 promoter with dCas9-TET. (A) Schematic of the murine Il33 genomic locus depicting the two transcriptional isoforms with a highlighted region of an 800bp region of the Il33-002 promoter and the locations of the 11 CpGs as well as 4 gRNAs targeting specific CpGs. The 11 CpGs are numbered sequentially in the 5’ to 3’ direction. The promoter-targeting gRNAs used in these experiments are shown relative to the CpGs and are approximately to scale such that CpGs 1, 2, and 3 are targeted by gRNA1, CpG 5 by gRNA 2, and gRNA 3 targets CpGs 9, 10, 11 – which overlap the transcription start site (TSS), marked by a black arrow. The orientation of the gRNAs is indicated by an arrow, where an arrow pointing to the left indicates a gRNA that binds the plus strand. (B) Percent of DNA methylation assayed by bisulfite-pyrosequencing at the three transcription start site (TSS) CpGs (labeled 9-11) following treatment of NIH-3T3 cells with indicated concentrations of 5-aza-2’-deoxycytidine or water control. (C) Expression of Il33-002 quantified by RT-qPCRand normalized to beta Actin ( Actb ) expression following treatment of NIH-3T3 cells with indicated concentrations of 5-aza-2’-deoxycytidine or water control. (D) Expression of predicted (Transfac) and experimentally validated (Qiagen, ENCODE, Gene Transcription Regulation Database) Il33-002 transcription factors quantified by RT-qPCR and normalized to Actb expression following treatment of NIH-3T3 cells with indicated concentrations of 5-aza-2’-deoxycytidine or water control. (E-G) Percent of DNA methylation assayed by bisulfite-pyrosequencing at 7 targeted CpGs in the Il33-002 promoter following transduction with lentiviruses and antibiotic selection of virally infected cells (gRNAs) or selection by flow cytometry (BFP; dCas9 constructs) of NIH-3T3 cells with dCas9-Tet/dCas9-deadTET (BFP) and gRNA1 (E), gRNA2 (F), or gRNA3 (G) compared to gRNAscr (light and dark grey, data identical in E-G and shown for comparison) (n=4-8). (H-I) Expression of Il33-002 (H) and Il33-001 (I) quantified by RT-qPCR and normalized to Actb expression in NIH-3T3 stably expressing one of 4 gRNAs and dCas9-TET or dCas9-deadTET. n=3 for all data panels, except E-G as indicated, and all data shown as (mean+/-SEM). (J) Relative light units normalized to protein quantity in transfected HEK293 cells. Cells were transiently transfected with methylated or unmethylated SV40-luciferase vector along with mammalian TET2 expression plasmid or empty vector (pcDNA3.1) control. * indicates statistically significant difference of p

    Journal: bioRxiv

    Article Title: Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

    doi: 10.1101/2020.03.28.012518

    Figure Lengend Snippet: Targeting the Il33 promoter with dCas9-TET. (A) Schematic of the murine Il33 genomic locus depicting the two transcriptional isoforms with a highlighted region of an 800bp region of the Il33-002 promoter and the locations of the 11 CpGs as well as 4 gRNAs targeting specific CpGs. The 11 CpGs are numbered sequentially in the 5’ to 3’ direction. The promoter-targeting gRNAs used in these experiments are shown relative to the CpGs and are approximately to scale such that CpGs 1, 2, and 3 are targeted by gRNA1, CpG 5 by gRNA 2, and gRNA 3 targets CpGs 9, 10, 11 – which overlap the transcription start site (TSS), marked by a black arrow. The orientation of the gRNAs is indicated by an arrow, where an arrow pointing to the left indicates a gRNA that binds the plus strand. (B) Percent of DNA methylation assayed by bisulfite-pyrosequencing at the three transcription start site (TSS) CpGs (labeled 9-11) following treatment of NIH-3T3 cells with indicated concentrations of 5-aza-2’-deoxycytidine or water control. (C) Expression of Il33-002 quantified by RT-qPCRand normalized to beta Actin ( Actb ) expression following treatment of NIH-3T3 cells with indicated concentrations of 5-aza-2’-deoxycytidine or water control. (D) Expression of predicted (Transfac) and experimentally validated (Qiagen, ENCODE, Gene Transcription Regulation Database) Il33-002 transcription factors quantified by RT-qPCR and normalized to Actb expression following treatment of NIH-3T3 cells with indicated concentrations of 5-aza-2’-deoxycytidine or water control. (E-G) Percent of DNA methylation assayed by bisulfite-pyrosequencing at 7 targeted CpGs in the Il33-002 promoter following transduction with lentiviruses and antibiotic selection of virally infected cells (gRNAs) or selection by flow cytometry (BFP; dCas9 constructs) of NIH-3T3 cells with dCas9-Tet/dCas9-deadTET (BFP) and gRNA1 (E), gRNA2 (F), or gRNA3 (G) compared to gRNAscr (light and dark grey, data identical in E-G and shown for comparison) (n=4-8). (H-I) Expression of Il33-002 (H) and Il33-001 (I) quantified by RT-qPCR and normalized to Actb expression in NIH-3T3 stably expressing one of 4 gRNAs and dCas9-TET or dCas9-deadTET. n=3 for all data panels, except E-G as indicated, and all data shown as (mean+/-SEM). (J) Relative light units normalized to protein quantity in transfected HEK293 cells. Cells were transiently transfected with methylated or unmethylated SV40-luciferase vector along with mammalian TET2 expression plasmid or empty vector (pcDNA3.1) control. * indicates statistically significant difference of p

    Article Snippet: Given that bound dCas9 envelopes nearly the entire DNA double helix [ ], we predicted that both CpG sites would be equally protected.

    Techniques: DNA Methylation Assay, Labeling, Expressing, Quantitative RT-PCR, Transduction, Selection, Infection, Flow Cytometry, Construct, Stable Transfection, Transfection, Methylation, Luciferase, Plasmid Preparation

    The effect of targeted promoter DNA demethylation on Il33 expression (A) Diagram illustrating the principle of site-specific demethylation with dCas9 removal in order to facilitate transcription factor binding to the newly demethylated region. First, DNA is endogenously methylated by DNMT1 with every round of replication and RNA-polII is not recruited to the promoter. After the introduction of dCas9 and a promoter-targeting gRNA, DNMT1 is physically occluded from the locus and nascent strands of DNA are unmethylated, facilitating passive demethylation of the bound region. However, RNA-polII is also physically occluded by dCas9. If dCas9 is successfully removed, the unmethylated DNA no longer serves as a substrate for DNMT1 and continues to remain unmethylated and RNA-polII may now be recruited. (B) Methylation of CpGs 9, 10, and 11 which had been previously demethylated by high-puromycin gRNA3:dCas9 in NIH-3T3 cells, after 75 days of passaging following the lentiviral transduction of Cre recombinase (pink) or empty-vector control (red). (C) Il33-002 expression in NIH-3T3 cell lines stably expressing gRNAscr (grey) or gRNA1 (blue), gRNA2 (purple), or gRNA3 (pink) under high-puromycin conditions in combination with dCas9, followed by dCas9 removal by Cre recombinase as assayed by RT-qPCR and normalized to Actb expression (n=3). (D-E) Il33-002 expression (D) or Il33-001 expression (E) in NIH-3T3 cells from (C) following treatment wither water control or 1 µM 5-aza-2’-deoxycytidine, measured by RT-qPCR and normalized to Actb expression (n=4-5). (F) Il33-002 expression measured by RT-qPCR and normalized to Actb expression, in dCas9:gRNAscr (grey) or dCas9:gRNA3 (pink) NIH-3T3 cells following Cre recombinase treatment and then treated with poly(I:C) (1 µg/mL) or water control for 4 or 8 hours. (G) DNA methylation assayed by bisulfite-pyrosequencing in NIH-3T3 cells expressing dCas9, gRNAscr, and Cre treated with 1 µg/mL poly(I:C) or water control for 8 hrs and 24 hrs (n=3). (H) Maximal Il33-002 induction (left y-axis, pink bars; data in log2 scale but axis numbering is not transformed) and maximal promoter demethylation (right y-axis, calculated as percent unmethylated divided by control methylation) under different treatments (x-axis: dCas9, 5-aza-2’-deoxycytidine, dCas9-VP64, dCas9-TET, and dCas9-deadTET). Where relevant, data for maximally inducing/demethylating gRNA is shown. * indicates statistically significant difference of p

    Journal: bioRxiv

    Article Title: Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

    doi: 10.1101/2020.03.28.012518

    Figure Lengend Snippet: The effect of targeted promoter DNA demethylation on Il33 expression (A) Diagram illustrating the principle of site-specific demethylation with dCas9 removal in order to facilitate transcription factor binding to the newly demethylated region. First, DNA is endogenously methylated by DNMT1 with every round of replication and RNA-polII is not recruited to the promoter. After the introduction of dCas9 and a promoter-targeting gRNA, DNMT1 is physically occluded from the locus and nascent strands of DNA are unmethylated, facilitating passive demethylation of the bound region. However, RNA-polII is also physically occluded by dCas9. If dCas9 is successfully removed, the unmethylated DNA no longer serves as a substrate for DNMT1 and continues to remain unmethylated and RNA-polII may now be recruited. (B) Methylation of CpGs 9, 10, and 11 which had been previously demethylated by high-puromycin gRNA3:dCas9 in NIH-3T3 cells, after 75 days of passaging following the lentiviral transduction of Cre recombinase (pink) or empty-vector control (red). (C) Il33-002 expression in NIH-3T3 cell lines stably expressing gRNAscr (grey) or gRNA1 (blue), gRNA2 (purple), or gRNA3 (pink) under high-puromycin conditions in combination with dCas9, followed by dCas9 removal by Cre recombinase as assayed by RT-qPCR and normalized to Actb expression (n=3). (D-E) Il33-002 expression (D) or Il33-001 expression (E) in NIH-3T3 cells from (C) following treatment wither water control or 1 µM 5-aza-2’-deoxycytidine, measured by RT-qPCR and normalized to Actb expression (n=4-5). (F) Il33-002 expression measured by RT-qPCR and normalized to Actb expression, in dCas9:gRNAscr (grey) or dCas9:gRNA3 (pink) NIH-3T3 cells following Cre recombinase treatment and then treated with poly(I:C) (1 µg/mL) or water control for 4 or 8 hours. (G) DNA methylation assayed by bisulfite-pyrosequencing in NIH-3T3 cells expressing dCas9, gRNAscr, and Cre treated with 1 µg/mL poly(I:C) or water control for 8 hrs and 24 hrs (n=3). (H) Maximal Il33-002 induction (left y-axis, pink bars; data in log2 scale but axis numbering is not transformed) and maximal promoter demethylation (right y-axis, calculated as percent unmethylated divided by control methylation) under different treatments (x-axis: dCas9, 5-aza-2’-deoxycytidine, dCas9-VP64, dCas9-TET, and dCas9-deadTET). Where relevant, data for maximally inducing/demethylating gRNA is shown. * indicates statistically significant difference of p

    Article Snippet: Given that bound dCas9 envelopes nearly the entire DNA double helix [ ], we predicted that both CpG sites would be equally protected.

    Techniques: Expressing, Binding Assay, Methylation, Passaging, Transduction, Plasmid Preparation, Stable Transfection, Quantitative RT-PCR, DNA Methylation Assay, Transformation Assay

    The effect of dCas9-based demethylation of TSS on expression of SerpinB5 , Tnf and FMR1 genes (A) (Top) Schematic of the human SERPINB5 promoter region, including the start site of transcription (marked by black arrow) and the binding site and PAM of the SERPINB5 gRNA. CG sequences are boxed in red. (Bottom) SERPINB5 gene with purple boxes indicating enhancer positions relative to gene body. Enhancer IDs correspond to the GeneHancer database. (B) DNA methylation level of each CpG averaged over 19 gRNAscr (red) and 23 gRNAmaspin (black) MDA-MB-231 clones as assessed by pyrosequencing (mean ± SEM). (C) Same data as (B) except now shown as the calculated methylation fraction for each of the 19 gRNAscr (red) and 23 gRNAmaspin (black) clones, rather than the average of all clones. (D) SERPINB5 expression levels measured by RT-qPCR and normalized to GAPDH expression levels for 5 gRNAscr and 5 lowly-methylated gRNAmaspin clones (mean ± SEM, n=5). (E) SERPINB5 expression levels measured by RT-qPCR and normalized to GAPDH expression levels for 48 (24 for each treatment) MDA-MB-231 clones subcloned from the clones in (D). (F) SERPINB5 expression levels measured by RT-qPCR and normalized to GAPDH expression levels for clones from (D) following treatment with 1 µM 5-aza-2’-deoxycytidine or water control (n=5). (G) Expression fold change of murine Il33-002 (grey) and Tnf (pink), normalized to Actb and water control, following treatment of control NIH-3T3 cells with 1 µM 5-aza-2’-deoxycytidine (n=3). (H) Tnf expression in NIH-3T3 cell lines stably in control (water); grey bars) or 1 µM 5-aza-2’-deoxycytidine (pink bars) expressing either gRNAscr or gRNA Tnf2 :Cre under high-puromycin conditions in combination with dCas9, followed by dCas9 removal by Cre recombinase, as assayed by RT-qPCR and normalized to Actb expression (n=3). (I) Schematic of the human FMR1 repeat region showing the 5’ untranslated region (UTR) that is prone to CGG repeat expansion and methylation in Fragile X syndrome. Sequence of the gRNA targeting this region is shown (gRNA-CGG) and the extent of the available binding sites for this gRNA is represented by purple lines which indicate binding sites, the 13 presented here represent less than 15% of the available binding site in the Fragile X syndrome patient primary fibroblasts used in this study, which have approximately 700 CGG repeats. (J) FMR1 expression quantified by RT-qPCR and normalized to GAPDH expression levels in Fragile X syndrome patient primary fibroblasts that had stably expressed dCas9 (later removed with Cre) and either gRNAscr (grey) or gRNA-CGG (purple) under high-puromycin selection (n=6). * indicates statistically significant difference of p

    Journal: bioRxiv

    Article Title: Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

    doi: 10.1101/2020.03.28.012518

    Figure Lengend Snippet: The effect of dCas9-based demethylation of TSS on expression of SerpinB5 , Tnf and FMR1 genes (A) (Top) Schematic of the human SERPINB5 promoter region, including the start site of transcription (marked by black arrow) and the binding site and PAM of the SERPINB5 gRNA. CG sequences are boxed in red. (Bottom) SERPINB5 gene with purple boxes indicating enhancer positions relative to gene body. Enhancer IDs correspond to the GeneHancer database. (B) DNA methylation level of each CpG averaged over 19 gRNAscr (red) and 23 gRNAmaspin (black) MDA-MB-231 clones as assessed by pyrosequencing (mean ± SEM). (C) Same data as (B) except now shown as the calculated methylation fraction for each of the 19 gRNAscr (red) and 23 gRNAmaspin (black) clones, rather than the average of all clones. (D) SERPINB5 expression levels measured by RT-qPCR and normalized to GAPDH expression levels for 5 gRNAscr and 5 lowly-methylated gRNAmaspin clones (mean ± SEM, n=5). (E) SERPINB5 expression levels measured by RT-qPCR and normalized to GAPDH expression levels for 48 (24 for each treatment) MDA-MB-231 clones subcloned from the clones in (D). (F) SERPINB5 expression levels measured by RT-qPCR and normalized to GAPDH expression levels for clones from (D) following treatment with 1 µM 5-aza-2’-deoxycytidine or water control (n=5). (G) Expression fold change of murine Il33-002 (grey) and Tnf (pink), normalized to Actb and water control, following treatment of control NIH-3T3 cells with 1 µM 5-aza-2’-deoxycytidine (n=3). (H) Tnf expression in NIH-3T3 cell lines stably in control (water); grey bars) or 1 µM 5-aza-2’-deoxycytidine (pink bars) expressing either gRNAscr or gRNA Tnf2 :Cre under high-puromycin conditions in combination with dCas9, followed by dCas9 removal by Cre recombinase, as assayed by RT-qPCR and normalized to Actb expression (n=3). (I) Schematic of the human FMR1 repeat region showing the 5’ untranslated region (UTR) that is prone to CGG repeat expansion and methylation in Fragile X syndrome. Sequence of the gRNA targeting this region is shown (gRNA-CGG) and the extent of the available binding sites for this gRNA is represented by purple lines which indicate binding sites, the 13 presented here represent less than 15% of the available binding site in the Fragile X syndrome patient primary fibroblasts used in this study, which have approximately 700 CGG repeats. (J) FMR1 expression quantified by RT-qPCR and normalized to GAPDH expression levels in Fragile X syndrome patient primary fibroblasts that had stably expressed dCas9 (later removed with Cre) and either gRNAscr (grey) or gRNA-CGG (purple) under high-puromycin selection (n=6). * indicates statistically significant difference of p

    Article Snippet: Given that bound dCas9 envelopes nearly the entire DNA double helix [ ], we predicted that both CpG sites would be equally protected.

    Techniques: Expressing, Binding Assay, DNA Methylation Assay, Multiple Displacement Amplification, Clone Assay, Methylation, Quantitative RT-PCR, Stable Transfection, Sequencing, Selection

    DNA methylation levels assessed by bisulfite-pyrosequencing of CpGs 1-6 in the SERPINB5 promoter in MDA-MB-231 cell lines stably expressing dCas9 and either gRNAscr (red) or gRNAmaspin (black), averaged across all 6 CpGs and plotted as a function of increasing puromycin concentration (n=1 per puromycin concentration).

    Journal: bioRxiv

    Article Title: Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

    doi: 10.1101/2020.03.28.012518

    Figure Lengend Snippet: DNA methylation levels assessed by bisulfite-pyrosequencing of CpGs 1-6 in the SERPINB5 promoter in MDA-MB-231 cell lines stably expressing dCas9 and either gRNAscr (red) or gRNAmaspin (black), averaged across all 6 CpGs and plotted as a function of increasing puromycin concentration (n=1 per puromycin concentration).

    Article Snippet: Given that bound dCas9 envelopes nearly the entire DNA double helix [ ], we predicted that both CpG sites would be equally protected.

    Techniques: DNA Methylation Assay, Multiple Displacement Amplification, Stable Transfection, Expressing, Concentration Assay

    Clinical observation of DNA quality index (DQI) in 211 consecutive surplus samples. The DNA quality of 211 consecutive surplus specimens-including 130 FFPE tissue blocks (FFPE Tissue), 27 FFPE cell blocks of FNA samples (FFPE FNA), 29 freshly centrifuged pleural fluid samples (fresh pleural fluid), and 25 FFPE cell blocks of pleural fluid (FFPE pleural fluid)-was evaluated. The DNA Quality Index and amplifiable DNA concentrations were determined using a DNA Quality Index Kit (Beijing ACCB Biotech Ltd., Beijing, China) according to the manufacturer’s instructions.

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Targeted next-generation sequencing in cytology specimens for molecular profiling of lung adenocarcinoma

    doi:

    Figure Lengend Snippet: Clinical observation of DNA quality index (DQI) in 211 consecutive surplus samples. The DNA quality of 211 consecutive surplus specimens-including 130 FFPE tissue blocks (FFPE Tissue), 27 FFPE cell blocks of FNA samples (FFPE FNA), 29 freshly centrifuged pleural fluid samples (fresh pleural fluid), and 25 FFPE cell blocks of pleural fluid (FFPE pleural fluid)-was evaluated. The DNA Quality Index and amplifiable DNA concentrations were determined using a DNA Quality Index Kit (Beijing ACCB Biotech Ltd., Beijing, China) according to the manufacturer’s instructions.

    Article Snippet: Genomic DNA was extracted from five consecutive 5-μm slides of formalin-fixed, paraffin-embedded (FFPE) cell blocks using a QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.

    Techniques: Formalin-fixed Paraffin-Embedded