lambda dna hindiii cut dna marker Search Results


λ phage dna  (New England Biolabs)
97
New England Biolabs λ phage dna
λ Phage Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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serum albumin  (Thermo Fisher)
99
Thermo Fisher serum albumin
Serum Albumin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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kir sso genotyping test  (Thermo Fisher)
90
Thermo Fisher kir sso genotyping test
Kir Sso Genotyping Test, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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bgt neb  (New England Biolabs)
96
New England Biolabs bgt neb
5mC and 5hmC detection by enzymatic deamination method. (A) Principle of the methodology: genomic DNA can either be treated with TET2 and <t>BGT</t> (left) to protect both 5mC and 5hmC or with BGT alone (right) to protect 5hmC. Subsequent deamination by APOBEC3A followed by PCR amplification allows the distinction between the unprotected substrate (read as T) from the protected cytosine derivatives (read as C). The TET2 and BGT treatment results in the distinction of 5mC and 5hmC from C, whereas BGT treatment results in the distinction of 5hmC from C and 5mC. (B) Deaminated cytosines from unmethylated lambda genome display no observable sequence preference by APOBEC(5mC) deamination method. (C) False-positive methylation calling rate (nonconversion error rate) of each cytosine dinucleotides sequence context (CpA [CA], CpC <t>[CC],</t> <t>CpG</t> [CG], and CpT [CT]) estimated from the unmethylated lambda genome for the enzymatic deamination method (APOBEC(5mC), two WGBS performed in this study, i.e., BS kit 1 and BS kit 2, and six published WGBS experiments sampled from the ENCODE Project) (Supplemental Table S3). (D) Deamination of 5mC in the fully methylated XP12 genome results in no observable sequence preference by APOBEC(5hmC) enzymatic deamination method. (E) Distribution patterns of 5mCpG (blue) and 5hmCpG (red: 50 ng library; pink: 1 ng library) at various protein/DNA interaction sites. The absolute (smooth lines) and normalized (dotted lines) 5hmC and 5mC levels in the CpG context are depicted around TET1, RNA polymerase II, and CTCF binding sites, as well as at active transcription chromatin mark (H3K4me3), repressive chromatin mark (H3K27me3), active enhancer mark (H3K27ac), and general enhancer (H3K4me1 in the absence of H3K4me3) regions. Unbound sites that are randomly sampled from the reference genome server as a control. (F) Pearson's correlation between 5hmC measured using sequencing of enzymatically deaminated DNA (x-axis) versus LC-MS (y-axis) for various genomic DNA. There are two technical replicates of the APOBEC(5hmC) sequencing method for each sample. 5hmC levels are presented as 1000 percentage, and both axes use the log scale.
Bgt Neb, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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lambda zapii cdna library synthesis kit  (Agilent technologies)
90
Agilent technologies lambda zapii cdna library synthesis kit
5mC and 5hmC detection by enzymatic deamination method. (A) Principle of the methodology: genomic DNA can either be treated with TET2 and <t>BGT</t> (left) to protect both 5mC and 5hmC or with BGT alone (right) to protect 5hmC. Subsequent deamination by APOBEC3A followed by PCR amplification allows the distinction between the unprotected substrate (read as T) from the protected cytosine derivatives (read as C). The TET2 and BGT treatment results in the distinction of 5mC and 5hmC from C, whereas BGT treatment results in the distinction of 5hmC from C and 5mC. (B) Deaminated cytosines from unmethylated lambda genome display no observable sequence preference by APOBEC(5mC) deamination method. (C) False-positive methylation calling rate (nonconversion error rate) of each cytosine dinucleotides sequence context (CpA [CA], CpC <t>[CC],</t> <t>CpG</t> [CG], and CpT [CT]) estimated from the unmethylated lambda genome for the enzymatic deamination method (APOBEC(5mC), two WGBS performed in this study, i.e., BS kit 1 and BS kit 2, and six published WGBS experiments sampled from the ENCODE Project) (Supplemental Table S3). (D) Deamination of 5mC in the fully methylated XP12 genome results in no observable sequence preference by APOBEC(5hmC) enzymatic deamination method. (E) Distribution patterns of 5mCpG (blue) and 5hmCpG (red: 50 ng library; pink: 1 ng library) at various protein/DNA interaction sites. The absolute (smooth lines) and normalized (dotted lines) 5hmC and 5mC levels in the CpG context are depicted around TET1, RNA polymerase II, and CTCF binding sites, as well as at active transcription chromatin mark (H3K4me3), repressive chromatin mark (H3K27me3), active enhancer mark (H3K27ac), and general enhancer (H3K4me1 in the absence of H3K4me3) regions. Unbound sites that are randomly sampled from the reference genome server as a control. (F) Pearson's correlation between 5hmC measured using sequencing of enzymatically deaminated DNA (x-axis) versus LC-MS (y-axis) for various genomic DNA. There are two technical replicates of the APOBEC(5hmC) sequencing method for each sample. 5hmC levels are presented as 1000 percentage, and both axes use the log scale.
Lambda Zapii Cdna Library Synthesis Kit, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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flowpra  (Thermo Fisher)
90
Thermo Fisher flowpra
5mC and 5hmC detection by enzymatic deamination method. (A) Principle of the methodology: genomic DNA can either be treated with TET2 and <t>BGT</t> (left) to protect both 5mC and 5hmC or with BGT alone (right) to protect 5hmC. Subsequent deamination by APOBEC3A followed by PCR amplification allows the distinction between the unprotected substrate (read as T) from the protected cytosine derivatives (read as C). The TET2 and BGT treatment results in the distinction of 5mC and 5hmC from C, whereas BGT treatment results in the distinction of 5hmC from C and 5mC. (B) Deaminated cytosines from unmethylated lambda genome display no observable sequence preference by APOBEC(5mC) deamination method. (C) False-positive methylation calling rate (nonconversion error rate) of each cytosine dinucleotides sequence context (CpA [CA], CpC <t>[CC],</t> <t>CpG</t> [CG], and CpT [CT]) estimated from the unmethylated lambda genome for the enzymatic deamination method (APOBEC(5mC), two WGBS performed in this study, i.e., BS kit 1 and BS kit 2, and six published WGBS experiments sampled from the ENCODE Project) (Supplemental Table S3). (D) Deamination of 5mC in the fully methylated XP12 genome results in no observable sequence preference by APOBEC(5hmC) enzymatic deamination method. (E) Distribution patterns of 5mCpG (blue) and 5hmCpG (red: 50 ng library; pink: 1 ng library) at various protein/DNA interaction sites. The absolute (smooth lines) and normalized (dotted lines) 5hmC and 5mC levels in the CpG context are depicted around TET1, RNA polymerase II, and CTCF binding sites, as well as at active transcription chromatin mark (H3K4me3), repressive chromatin mark (H3K27me3), active enhancer mark (H3K27ac), and general enhancer (H3K4me1 in the absence of H3K4me3) regions. Unbound sites that are randomly sampled from the reference genome server as a control. (F) Pearson's correlation between 5hmC measured using sequencing of enzymatically deaminated DNA (x-axis) versus LC-MS (y-axis) for various genomic DNA. There are two technical replicates of the APOBEC(5hmC) sequencing method for each sample. 5hmC levels are presented as 1000 percentage, and both axes use the log scale.
Flowpra, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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hla fusion software v4.2  (Thermo Fisher)
90
Thermo Fisher hla fusion software v4.2
5mC and 5hmC detection by enzymatic deamination method. (A) Principle of the methodology: genomic DNA can either be treated with TET2 and <t>BGT</t> (left) to protect both 5mC and 5hmC or with BGT alone (right) to protect 5hmC. Subsequent deamination by APOBEC3A followed by PCR amplification allows the distinction between the unprotected substrate (read as T) from the protected cytosine derivatives (read as C). The TET2 and BGT treatment results in the distinction of 5mC and 5hmC from C, whereas BGT treatment results in the distinction of 5hmC from C and 5mC. (B) Deaminated cytosines from unmethylated lambda genome display no observable sequence preference by APOBEC(5mC) deamination method. (C) False-positive methylation calling rate (nonconversion error rate) of each cytosine dinucleotides sequence context (CpA [CA], CpC <t>[CC],</t> <t>CpG</t> [CG], and CpT [CT]) estimated from the unmethylated lambda genome for the enzymatic deamination method (APOBEC(5mC), two WGBS performed in this study, i.e., BS kit 1 and BS kit 2, and six published WGBS experiments sampled from the ENCODE Project) (Supplemental Table S3). (D) Deamination of 5mC in the fully methylated XP12 genome results in no observable sequence preference by APOBEC(5hmC) enzymatic deamination method. (E) Distribution patterns of 5mCpG (blue) and 5hmCpG (red: 50 ng library; pink: 1 ng library) at various protein/DNA interaction sites. The absolute (smooth lines) and normalized (dotted lines) 5hmC and 5mC levels in the CpG context are depicted around TET1, RNA polymerase II, and CTCF binding sites, as well as at active transcription chromatin mark (H3K4me3), repressive chromatin mark (H3K27me3), active enhancer mark (H3K27ac), and general enhancer (H3K4me1 in the absence of H3K4me3) regions. Unbound sites that are randomly sampled from the reference genome server as a control. (F) Pearson's correlation between 5hmC measured using sequencing of enzymatically deaminated DNA (x-axis) versus LC-MS (y-axis) for various genomic DNA. There are two technical replicates of the APOBEC(5hmC) sequencing method for each sample. 5hmC levels are presented as 1000 percentage, and both axes use the log scale.
Hla Fusion Software V4.2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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typestream visual ngs analysis software  (Thermo Fisher)
90
Thermo Fisher typestream visual ngs analysis software
5mC and 5hmC detection by enzymatic deamination method. (A) Principle of the methodology: genomic DNA can either be treated with TET2 and <t>BGT</t> (left) to protect both 5mC and 5hmC or with BGT alone (right) to protect 5hmC. Subsequent deamination by APOBEC3A followed by PCR amplification allows the distinction between the unprotected substrate (read as T) from the protected cytosine derivatives (read as C). The TET2 and BGT treatment results in the distinction of 5mC and 5hmC from C, whereas BGT treatment results in the distinction of 5hmC from C and 5mC. (B) Deaminated cytosines from unmethylated lambda genome display no observable sequence preference by APOBEC(5mC) deamination method. (C) False-positive methylation calling rate (nonconversion error rate) of each cytosine dinucleotides sequence context (CpA [CA], CpC <t>[CC],</t> <t>CpG</t> [CG], and CpT [CT]) estimated from the unmethylated lambda genome for the enzymatic deamination method (APOBEC(5mC), two WGBS performed in this study, i.e., BS kit 1 and BS kit 2, and six published WGBS experiments sampled from the ENCODE Project) (Supplemental Table S3). (D) Deamination of 5mC in the fully methylated XP12 genome results in no observable sequence preference by APOBEC(5hmC) enzymatic deamination method. (E) Distribution patterns of 5mCpG (blue) and 5hmCpG (red: 50 ng library; pink: 1 ng library) at various protein/DNA interaction sites. The absolute (smooth lines) and normalized (dotted lines) 5hmC and 5mC levels in the CpG context are depicted around TET1, RNA polymerase II, and CTCF binding sites, as well as at active transcription chromatin mark (H3K4me3), repressive chromatin mark (H3K27me3), active enhancer mark (H3K27ac), and general enhancer (H3K4me1 in the absence of H3K4me3) regions. Unbound sites that are randomly sampled from the reference genome server as a control. (F) Pearson's correlation between 5hmC measured using sequencing of enzymatically deaminated DNA (x-axis) versus LC-MS (y-axis) for various genomic DNA. There are two technical replicates of the APOBEC(5hmC) sequencing method for each sample. 5hmC levels are presented as 1000 percentage, and both axes use the log scale.
Typestream Visual Ngs Analysis Software, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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hla fusion software  (Thermo Fisher)
90
Thermo Fisher hla fusion software
5mC and 5hmC detection by enzymatic deamination method. (A) Principle of the methodology: genomic DNA can either be treated with TET2 and <t>BGT</t> (left) to protect both 5mC and 5hmC or with BGT alone (right) to protect 5hmC. Subsequent deamination by APOBEC3A followed by PCR amplification allows the distinction between the unprotected substrate (read as T) from the protected cytosine derivatives (read as C). The TET2 and BGT treatment results in the distinction of 5mC and 5hmC from C, whereas BGT treatment results in the distinction of 5hmC from C and 5mC. (B) Deaminated cytosines from unmethylated lambda genome display no observable sequence preference by APOBEC(5mC) deamination method. (C) False-positive methylation calling rate (nonconversion error rate) of each cytosine dinucleotides sequence context (CpA [CA], CpC <t>[CC],</t> <t>CpG</t> [CG], and CpT [CT]) estimated from the unmethylated lambda genome for the enzymatic deamination method (APOBEC(5mC), two WGBS performed in this study, i.e., BS kit 1 and BS kit 2, and six published WGBS experiments sampled from the ENCODE Project) (Supplemental Table S3). (D) Deamination of 5mC in the fully methylated XP12 genome results in no observable sequence preference by APOBEC(5hmC) enzymatic deamination method. (E) Distribution patterns of 5mCpG (blue) and 5hmCpG (red: 50 ng library; pink: 1 ng library) at various protein/DNA interaction sites. The absolute (smooth lines) and normalized (dotted lines) 5hmC and 5mC levels in the CpG context are depicted around TET1, RNA polymerase II, and CTCF binding sites, as well as at active transcription chromatin mark (H3K4me3), repressive chromatin mark (H3K27me3), active enhancer mark (H3K27ac), and general enhancer (H3K4me1 in the absence of H3K4me3) regions. Unbound sites that are randomly sampled from the reference genome server as a control. (F) Pearson's correlation between 5hmC measured using sequencing of enzymatically deaminated DNA (x-axis) versus LC-MS (y-axis) for various genomic DNA. There are two technical replicates of the APOBEC(5hmC) sequencing method for each sample. 5hmC levels are presented as 1000 percentage, and both axes use the log scale.
Hla Fusion Software, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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labscreentm mixed class & ii  (Thermo Fisher)
90
Thermo Fisher labscreentm mixed class & ii
5mC and 5hmC detection by enzymatic deamination method. (A) Principle of the methodology: genomic DNA can either be treated with TET2 and <t>BGT</t> (left) to protect both 5mC and 5hmC or with BGT alone (right) to protect 5hmC. Subsequent deamination by APOBEC3A followed by PCR amplification allows the distinction between the unprotected substrate (read as T) from the protected cytosine derivatives (read as C). The TET2 and BGT treatment results in the distinction of 5mC and 5hmC from C, whereas BGT treatment results in the distinction of 5hmC from C and 5mC. (B) Deaminated cytosines from unmethylated lambda genome display no observable sequence preference by APOBEC(5mC) deamination method. (C) False-positive methylation calling rate (nonconversion error rate) of each cytosine dinucleotides sequence context (CpA [CA], CpC <t>[CC],</t> <t>CpG</t> [CG], and CpT [CT]) estimated from the unmethylated lambda genome for the enzymatic deamination method (APOBEC(5mC), two WGBS performed in this study, i.e., BS kit 1 and BS kit 2, and six published WGBS experiments sampled from the ENCODE Project) (Supplemental Table S3). (D) Deamination of 5mC in the fully methylated XP12 genome results in no observable sequence preference by APOBEC(5hmC) enzymatic deamination method. (E) Distribution patterns of 5mCpG (blue) and 5hmCpG (red: 50 ng library; pink: 1 ng library) at various protein/DNA interaction sites. The absolute (smooth lines) and normalized (dotted lines) 5hmC and 5mC levels in the CpG context are depicted around TET1, RNA polymerase II, and CTCF binding sites, as well as at active transcription chromatin mark (H3K4me3), repressive chromatin mark (H3K27me3), active enhancer mark (H3K27ac), and general enhancer (H3K4me1 in the absence of H3K4me3) regions. Unbound sites that are randomly sampled from the reference genome server as a control. (F) Pearson's correlation between 5hmC measured using sequencing of enzymatically deaminated DNA (x-axis) versus LC-MS (y-axis) for various genomic DNA. There are two technical replicates of the APOBEC(5hmC) sequencing method for each sample. 5hmC levels are presented as 1000 percentage, and both axes use the log scale.
Labscreentm Mixed Class & Ii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1x t4 ligase buffer  (New England Biolabs)
96
New England Biolabs 1x t4 ligase buffer
5mC and 5hmC detection by enzymatic deamination method. (A) Principle of the methodology: genomic DNA can either be treated with TET2 and <t>BGT</t> (left) to protect both 5mC and 5hmC or with BGT alone (right) to protect 5hmC. Subsequent deamination by APOBEC3A followed by PCR amplification allows the distinction between the unprotected substrate (read as T) from the protected cytosine derivatives (read as C). The TET2 and BGT treatment results in the distinction of 5mC and 5hmC from C, whereas BGT treatment results in the distinction of 5hmC from C and 5mC. (B) Deaminated cytosines from unmethylated lambda genome display no observable sequence preference by APOBEC(5mC) deamination method. (C) False-positive methylation calling rate (nonconversion error rate) of each cytosine dinucleotides sequence context (CpA [CA], CpC <t>[CC],</t> <t>CpG</t> [CG], and CpT [CT]) estimated from the unmethylated lambda genome for the enzymatic deamination method (APOBEC(5mC), two WGBS performed in this study, i.e., BS kit 1 and BS kit 2, and six published WGBS experiments sampled from the ENCODE Project) (Supplemental Table S3). (D) Deamination of 5mC in the fully methylated XP12 genome results in no observable sequence preference by APOBEC(5hmC) enzymatic deamination method. (E) Distribution patterns of 5mCpG (blue) and 5hmCpG (red: 50 ng library; pink: 1 ng library) at various protein/DNA interaction sites. The absolute (smooth lines) and normalized (dotted lines) 5hmC and 5mC levels in the CpG context are depicted around TET1, RNA polymerase II, and CTCF binding sites, as well as at active transcription chromatin mark (H3K4me3), repressive chromatin mark (H3K27me3), active enhancer mark (H3K27ac), and general enhancer (H3K4me1 in the absence of H3K4me3) regions. Unbound sites that are randomly sampled from the reference genome server as a control. (F) Pearson's correlation between 5hmC measured using sequencing of enzymatically deaminated DNA (x-axis) versus LC-MS (y-axis) for various genomic DNA. There are two technical replicates of the APOBEC(5hmC) sequencing method for each sample. 5hmC levels are presented as 1000 percentage, and both axes use the log scale.
1x T4 Ligase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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hind iii digested λ dna  (New England Biolabs)
93
New England Biolabs hind iii digested λ dna
5mC and 5hmC detection by enzymatic deamination method. (A) Principle of the methodology: genomic DNA can either be treated with TET2 and <t>BGT</t> (left) to protect both 5mC and 5hmC or with BGT alone (right) to protect 5hmC. Subsequent deamination by APOBEC3A followed by PCR amplification allows the distinction between the unprotected substrate (read as T) from the protected cytosine derivatives (read as C). The TET2 and BGT treatment results in the distinction of 5mC and 5hmC from C, whereas BGT treatment results in the distinction of 5hmC from C and 5mC. (B) Deaminated cytosines from unmethylated lambda genome display no observable sequence preference by APOBEC(5mC) deamination method. (C) False-positive methylation calling rate (nonconversion error rate) of each cytosine dinucleotides sequence context (CpA [CA], CpC <t>[CC],</t> <t>CpG</t> [CG], and CpT [CT]) estimated from the unmethylated lambda genome for the enzymatic deamination method (APOBEC(5mC), two WGBS performed in this study, i.e., BS kit 1 and BS kit 2, and six published WGBS experiments sampled from the ENCODE Project) (Supplemental Table S3). (D) Deamination of 5mC in the fully methylated XP12 genome results in no observable sequence preference by APOBEC(5hmC) enzymatic deamination method. (E) Distribution patterns of 5mCpG (blue) and 5hmCpG (red: 50 ng library; pink: 1 ng library) at various protein/DNA interaction sites. The absolute (smooth lines) and normalized (dotted lines) 5hmC and 5mC levels in the CpG context are depicted around TET1, RNA polymerase II, and CTCF binding sites, as well as at active transcription chromatin mark (H3K4me3), repressive chromatin mark (H3K27me3), active enhancer mark (H3K27ac), and general enhancer (H3K4me1 in the absence of H3K4me3) regions. Unbound sites that are randomly sampled from the reference genome server as a control. (F) Pearson's correlation between 5hmC measured using sequencing of enzymatically deaminated DNA (x-axis) versus LC-MS (y-axis) for various genomic DNA. There are two technical replicates of the APOBEC(5hmC) sequencing method for each sample. 5hmC levels are presented as 1000 percentage, and both axes use the log scale.
Hind Iii Digested λ Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


5mC and 5hmC detection by enzymatic deamination method. (A) Principle of the methodology: genomic DNA can either be treated with TET2 and BGT (left) to protect both 5mC and 5hmC or with BGT alone (right) to protect 5hmC. Subsequent deamination by APOBEC3A followed by PCR amplification allows the distinction between the unprotected substrate (read as T) from the protected cytosine derivatives (read as C). The TET2 and BGT treatment results in the distinction of 5mC and 5hmC from C, whereas BGT treatment results in the distinction of 5hmC from C and 5mC. (B) Deaminated cytosines from unmethylated lambda genome display no observable sequence preference by APOBEC(5mC) deamination method. (C) False-positive methylation calling rate (nonconversion error rate) of each cytosine dinucleotides sequence context (CpA [CA], CpC [CC], CpG [CG], and CpT [CT]) estimated from the unmethylated lambda genome for the enzymatic deamination method (APOBEC(5mC), two WGBS performed in this study, i.e., BS kit 1 and BS kit 2, and six published WGBS experiments sampled from the ENCODE Project) (Supplemental Table S3). (D) Deamination of 5mC in the fully methylated XP12 genome results in no observable sequence preference by APOBEC(5hmC) enzymatic deamination method. (E) Distribution patterns of 5mCpG (blue) and 5hmCpG (red: 50 ng library; pink: 1 ng library) at various protein/DNA interaction sites. The absolute (smooth lines) and normalized (dotted lines) 5hmC and 5mC levels in the CpG context are depicted around TET1, RNA polymerase II, and CTCF binding sites, as well as at active transcription chromatin mark (H3K4me3), repressive chromatin mark (H3K27me3), active enhancer mark (H3K27ac), and general enhancer (H3K4me1 in the absence of H3K4me3) regions. Unbound sites that are randomly sampled from the reference genome server as a control. (F) Pearson's correlation between 5hmC measured using sequencing of enzymatically deaminated DNA (x-axis) versus LC-MS (y-axis) for various genomic DNA. There are two technical replicates of the APOBEC(5hmC) sequencing method for each sample. 5hmC levels are presented as 1000 percentage, and both axes use the log scale.

Journal: Genome Research

Article Title: Nondestructive enzymatic deamination enables single-molecule long-read amplicon sequencing for the determination of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution

doi: 10.1101/gr.265306.120

Figure Lengend Snippet: 5mC and 5hmC detection by enzymatic deamination method. (A) Principle of the methodology: genomic DNA can either be treated with TET2 and BGT (left) to protect both 5mC and 5hmC or with BGT alone (right) to protect 5hmC. Subsequent deamination by APOBEC3A followed by PCR amplification allows the distinction between the unprotected substrate (read as T) from the protected cytosine derivatives (read as C). The TET2 and BGT treatment results in the distinction of 5mC and 5hmC from C, whereas BGT treatment results in the distinction of 5hmC from C and 5mC. (B) Deaminated cytosines from unmethylated lambda genome display no observable sequence preference by APOBEC(5mC) deamination method. (C) False-positive methylation calling rate (nonconversion error rate) of each cytosine dinucleotides sequence context (CpA [CA], CpC [CC], CpG [CG], and CpT [CT]) estimated from the unmethylated lambda genome for the enzymatic deamination method (APOBEC(5mC), two WGBS performed in this study, i.e., BS kit 1 and BS kit 2, and six published WGBS experiments sampled from the ENCODE Project) (Supplemental Table S3). (D) Deamination of 5mC in the fully methylated XP12 genome results in no observable sequence preference by APOBEC(5hmC) enzymatic deamination method. (E) Distribution patterns of 5mCpG (blue) and 5hmCpG (red: 50 ng library; pink: 1 ng library) at various protein/DNA interaction sites. The absolute (smooth lines) and normalized (dotted lines) 5hmC and 5mC levels in the CpG context are depicted around TET1, RNA polymerase II, and CTCF binding sites, as well as at active transcription chromatin mark (H3K4me3), repressive chromatin mark (H3K27me3), active enhancer mark (H3K27ac), and general enhancer (H3K4me1 in the absence of H3K4me3) regions. Unbound sites that are randomly sampled from the reference genome server as a control. (F) Pearson's correlation between 5hmC measured using sequencing of enzymatically deaminated DNA (x-axis) versus LC-MS (y-axis) for various genomic DNA. There are two technical replicates of the APOBEC(5hmC) sequencing method for each sample. 5hmC levels are presented as 1000 percentage, and both axes use the log scale.

Article Snippet: Enzymatic deamination for 5hmC detection For 5hmC detection, 200 ng of mouse E14 genomic DNA was mixed with 10 ng unmethylated lambda DNA, 10 ng of T4gt phage DNA, and 1 ng of CpG methylated pUC19 DNA and then incubated with 20 U of BGT (NEB) in 1× NEBuffer 2 for 2 h at 37°C.

Techniques: Amplification, Sequencing, Methylation, Binding Assay, Control, Liquid Chromatography with Mass Spectroscopy