Bisulfite sequencing was performed as described previously (12 (link)). Briefly, genomic DNA (about 3 μg) from five replicates of 35-DAF endosperm and embryo from the cross ZB107×ZB306 was isolated using the Plant DNeasy Minikit (Qiagen, Valencia, CA, USA), and then was shared by sonication to fragments of 300–500 bp. Custom Illumina adapters were ligated following the manufacturer's protocols. Fragments with adapters were bisulfite converted twice by sodium bisulfite using EpiTech Bisulfite Kits (Qiagen, Valencia, CA, USA) and then amplified by 18 cycles of polymerase chain reaction (PCR) using Pfu DNA polymerase (TaKaRa, Dalian, China). Following this process, the libraries were sequenced at BGI (Shenzhen), generating paired-end 90 bp reads from each library.
Epitech bisulfite kit
Manufactured by Qiagen
Sourced in United States, Germany
The EpiTech Bisulfite Kit is a product designed for the conversion of DNA samples through bisulfite treatment. This process is a crucial step in epigenetic studies, enabling the analysis of DNA methylation patterns. The kit provides the necessary reagents and protocol to perform this conversion efficiently.
Automatically generated - may contain errors
Lab products found in correlation
Pyromark pcr kit, by Qiagen (7 mentions)
Pyromark gold q24 reagent, by Qiagen (5 mentions)
Allprep dna rna micro kit, by Qiagen (4 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Dneasy plant mini kit, by Qiagen (2 mentions)
Qiaamp dna blood mini kit, by Qiagen (2 mentions)
Control methylated dna, by Qiagen (2 mentions)
Unmethylated dna, by Qiagen (2 mentions)
Pgem t easy vector system 1, by Promega (2 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
Pyromark q24, by Qiagen (2 mentions)
Ta cloning kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Pfu dna polymerase, by Takara Bio (1 mentions)
Pyromark assay design software, by Qiagen (1 mentions)
Qiaprep spin miniprep kit, by Qiagen (1 mentions)
Minibest agarose gel dna extraction kit, by Takara Bio (1 mentions)
Mastercycler gradient, by Eppendorf (1 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Methyl primer express software v1, by Thermo Fisher Scientific (1 mentions)
45 protocols using epitech bisulfite kit
1
Whole-genome Bisulfite Sequencing of Maize
2
Quantitative DNA Methylation Analysis
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Human blood genomic DNA was extracted and quantified as described previously (31 (link)). The DNA methylation assay comprised of sodium bisulfite DNA conversion (EpiTech Bisulfite kits; Qiagen AB, Sollentuna, Sweden), polymerase chain reaction (PCR) amplification (Pyromark PCR kit; Qiagen) and pyrosequencing (Pyromark Gold Q24 Reagents; Qiagen AB), which were performed in accordance with the manufacturer's protocol. The Pyromark PCR Master Mix was used in PCR amplification. PCR primers were designed using PyroMark Assay Design software v2.0.1.15 (both Qiagen). The sequences of the primers utilized were as follows: CDKN2B forward, 5′-TAGGGGGAGGAGTTTAAGGGG-3′ and reverse, 5′-biotin-ACACTCTTCCCTTCTTTCC-3′; CDKN2B sequencing primer; 5′-GGGGTAGTGAGGATT-3′. The thermocycling conditions were as follows: 1 cycle at 94°C for 15 sex, 45 cycles at 94°C for 20 sec, 58°C for 30 sec, 72°C for 60 sec and an extension stage at 72°C for 3 min.
3
Nucleic Acid Extraction and Methylation Analysis
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Nucleic acid extraction analyzer (Lab-Aid 820, Xiamen, China) was used to extract genomic DNA from peripheral blood samples. The concentrations of extracted DNA were measured by the ultramicro nucleic acid ultraviolet tester (NANODROP 1000, Wilmington, USA). Plasma levels of biochemical factors (including TG, TC, HDL, LDL, ApoA 1, ApoB, ApoE, Lp(a), hs-CRP, ALB, GLB, ALT, AST, ALP and GGT) were measured using the methods described in our previous study [30 ]. DNA methylation was measured using the sodium bisulphite DNA conversion coupled with pyrosequencing [30 ]. Genomic DNA was chemically modified by sodium bisulfite (EpiTech Bisulfite Kits; Qiagen) to convert all unmethylated cytosines to uracils while the methylated cytosines unchanged. The bisulfite converted DNA and the polymerase chain reaction (PCR) primers which were designed by PyroMark Assay Design software were mixed and performed with PCR (Pyromark PCR Kit; Qiagen). The PCR products were degenerated and released to single strand products for pyrosequencing and the sequencing was conducted by Q24 machine and reagents (Pyromark Gold Q24 Reagents; Qiagen). The forward primer sequence was 5′-TGGATGGTTTAGTGTATAAGTTGTATT-3′. The reverse primer sequence was 5′-Biotin-CACCTCATCCTCCACATTCAT-3′, and the sequencing primer sequence was 5′-AAGTGGGGTTTAAAAAG-3′.
4
DNA Methylation Analysis of P2Y12 Gene
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Human genomic DNA was extracted from leucocytes of peripheral blood samples with a commercially available kit (QIAamp DNA Blood Mini Kit, Qiagen, Hilden, Germany). DNA concentrations were quantified by the ultramicronucleic acid ultraviolet tester (NANODROP 1000, Wilmington, USA), and all of them were more than 500 ng/μL.
Bisulfite pyrosequencing technology was applied to determine the methylation levels of 2 CpG dinucleotides on the fragment of P2Y12 gene promoter, which combines with sodium bisulfite DNA conversion chemistry (EpiTech Bisulfite Kits; Qiagen), polymerase chain reaction (PCR) amplification (Pyromark PCR Kit; Qiagen), and sequencing by synthesis assay (Pyromark Gold Q24 Reagents; Qiagen) of the target sequence. We selected PyroMark Assay Design software for planning PCR primers. Sequences of the PCR and pyrosequencing primers were described inTable 1 .
Bisulfite pyrosequencing technology was applied to determine the methylation levels of 2 CpG dinucleotides on the fragment of P2Y12 gene promoter, which combines with sodium bisulfite DNA conversion chemistry (EpiTech Bisulfite Kits; Qiagen), polymerase chain reaction (PCR) amplification (Pyromark PCR Kit; Qiagen), and sequencing by synthesis assay (Pyromark Gold Q24 Reagents; Qiagen) of the target sequence. We selected PyroMark Assay Design software for planning PCR primers. Sequences of the PCR and pyrosequencing primers were described in
5
Bisulfite Conversion for DNA Methylation
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Genomic DNA from the NAc was extracted using the triple prep kit from QIAgene according to the manufacturer’s instruction. DNA was sent to the Innovation Center of Genome Quebec where it was treated with sodium bisulfite (Na-BIS) using the Epitech Bisulfite kit (QIAgen) and where Epityper40 (link) was performed.
6
Quantification of PON1 Gene Methylation
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The QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) was used to extract human genomic DNA from the leukocytes of peripheral blood samples. The DNA concentrations, which must be greater than 500 ng/μL, were quantified using the NanoDrop 1000 (NanoDrop, Wilmington, DE). The levels of DNA methylation in 4 CpG dinucleotides, which were located on the PON1 gene promoter (GRCh37.p13:94954884‐94952884) (Figure S1 ), were determined using bisulfite pyrosequencing technology. The process of bisulfite pyrosequencing included sodium bisulfite DNA conversion chemistry using the EpiTech Bisulfite Kit (Qiagen, Valencia, CA), polymerase chain reaction (PCR) amplification using the PyroMark PCR Kit, as well as the targeted fragment sequencing using PyroMark Gold Q24 Reagents.19 Through PyroMark Assay Design software, the PCR and pyrosequencing primers were designed, and each of them is listed in Table S1 .
7
DNA Methylation Profiling of CD4+ T Cells
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Genomic DNA was isolated and purified from CD4+ T cells using an AllPrep DNA/RNA Micro kit (Qiagen GmbH, Hilden, Germany). Sodium bisulfite conversion bisulfite-sequencing PCR was performed using an EpiTech Bisulfite kit (Qiagen GmbH, Hilden, Germany). PCR products were subcloned into a pTZ57R/T vector (InsTAcloneTM PCR Cloning kit, Fermentas Inc., Ontario, Canada). Ligated vectors were then transferred into the Escherichia coli (E. coli) strains DH5α and ten positive colonies were extracted by Qiaprep® Spin Miniprep kit (Qiagen GmbH, Hilden, Germany) and sequenced. The sequences were analyzed with bisulfite sequencing DNA methylation analysis (BISMA) online software.
8
Genomic DNA Extraction and Bisulfite Sequencing from Chicken Spermatozoa
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Genomic DNA was extracted and purified from chicken spermatozoa using an AllPrep DNA/RNA Micro Kit (QIAGEN) following the manufacturer’s instructions as previously described by Kito, et al.68 (link). Sodium bisulfite conversion was performed using 1 μl of genomic DNA (1 μg/μl), 19 μl of RNase-free water, 85 μl of Bisulfite mix, and 35 μl of DNA protect buffer with a Master Cycler Gradient (Eppendorf, Hamburg, Germany) following to the manufacturer’s instructions of the EpiTech Bisulfite Kit (QIAGEN). Bisulfite-sequencing PCR (BSP) amplification was performed using 1 μl of purified bisulfite-converted DNA (50 ng/μl), 10 μl of 2× Prime Taq Premix, 1 μl of upstream primer (5 pmol/μl), 1 μl of downstream primer (5 pmol/μl), and 7 μl of DEPC-treated water. Primers for BSP were designed using the MethPrimer (http://www.urogene.org/methprimer/ ) (see Supplementary Table S4 ). BSP amplification products were purified using the MiniBest Agarose Gel DNA Extraction Kit (TaKaRa Bio Inc., Kusatsu, Shiga, Japan) following the manufacturer’s instructions.
9
Bisulfite Sequencing of CGI Methylation
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Npdc1 and Smc6l promoter CpG island (CGI) methylation analyses were performed by bisulfite sequencing using the cloning and Sanger sequencing approach. A total of 2 μg of genomic DNA was treated with sodium bisulfite using the EpiTech Bisulfite Kit (Qiagen, MD, USA). PCR primers were designed by Methyl Primer Express software v1.0 (Applied Biosystems, CA;supplementary Table S8, Supplementary Material online). The amplicons were cloned using Topo TA Cloning Kit (Life Technologies, CA, USA). Transformed colonies were selected and sequenced using M13 forward primer at Beckman Coulter Genomics (Danvers, MA, USA). The sequences were then analyzed using Sequencher 4.10.
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Npdc1 and Smc6l promoter CpG island (CGI) methylation analyses were performed by bisulfite sequencing using the cloning and Sanger sequencing approach. A total of 2 μg of genomic DNA was treated with sodium bisulfite using the EpiTech Bisulfite Kit (Qiagen, MD, USA). PCR primers were designed by Methyl Primer Express software v1.0 (Applied Biosystems, CA;
10
Epigenetic Profiling of Pluripotent Stem Cells
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The genomic DNA from human fibroblasts, H9-hESCs, and iPS cells was extracted using an Exgene Tissue SV kit (GeneAll Biotechnology, Seoul, Korea), and the DNAs were treated with an EpiTech Bisulfite Kit (Qiagen GmbH, Hilden, Germany) for bisulfite conversion according to the manufacturer's instructions. The “CpG-rich” promoter regions of the human Oct4 and Nanog genes were amplified using the specific PCR primers listed in S1 Table. The amplified PCR products were subcloned into the TA cloning vector (RBC Bioscience Corp., New Taipei City, Taiwan) and were subjected to sequencing analysis.
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