Detect-seq experiment was conducted as reported previously18 (link). About 4 × 105 HEK293T cells were plated on a six-well plate and 1250 ng of each DdCBE monomer was used for transfection. Cells were harvested 72 h after transfection, and genomic DNA was isolated using DNA Isolation Mini Kit (Vazyme, DC112). DNA damage was repaired or protected to reduce the background noise. DNA fragments containing DdCBE-induced deoxyuridine bases were recognized by UDG and labeled via nick translation and subsequent chemical reactions. The biotin-labeled fragments were enriched by streptavidin C1 beads (Invitrogen, 65001) and ligated with Y adapters. Detect-seq libraries were sequenced on MGISEQ-2000. The efficiency and specificity of Detect-seq libraries were evaluated by qPCR and Sanger sequencing on spike-in molecules. Detect-seq data were mapped and processed following the steps described in the Detect-seq tool website (www.detect-seq.com ). The DdCBE default parameters were used for analysis.
Dna isolation mini kit
Manufactured by Vazyme
Sourced in China
The DNA Isolation Mini Kit is a laboratory tool designed to efficiently extract and purify DNA from various sample types. It utilizes a rapid and reliable method to isolate high-quality DNA, suitable for downstream applications such as PCR, sequencing, and molecular analysis.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin c1 beads, by Thermo Fisher Scientific (1 mentions)
Paris kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Mirneasy serum plasma kit, by Qiagen (1 mentions)
Pcr master mix, by Vazyme (1 mentions)
Lightcycler 480, by Roche (1 mentions)
5 protocols using dna isolation mini kit
1
Detect-seq for Mapping DdCBE-induced DNA Edits
2
Isolating RNA and DNA from ESCC
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Total RNA was purified from ESCC tissues and cells using TRIzol reagent (Invitrogen, Waltham, MA) and plasma using a miRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany). RNA in nuclear and cytoplasmic fractions was isolated using a PARIS kit (Thermo Fisher Scientific, Waltham, MA). Genomic DNA was extracted from cells using the DNA Isolation Mini Kit (Vazyme Biotech Co., Ltd, Nanjing, China).
3
Measuring Mitochondrial DNA Copy Number
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For this step, we followed the method referred to in our previous research [43 (link)]. Total cellular DNA was extracted using the DNA Isolation Mini Kit (DC102-01, Vazyme, Nanjing, Jiangsu, China) according to the manufacturer’s instructions and used for the detection of mtDNA copy number by qPCR with the following reagents: TaqMan Universal PCR master mix and the primers (16S rRNA and 18S rRNA). MtDNA levels were assessed using the mitochondrial genes 16S rRNA; nuclear 18S rRNA served as a loading control.
4
Mouse Tail DNA Extraction and SSLP Analysis
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The SSLP determination was performed as previously described (Zhou et al., 2001 (link)). Sequences for the primer pairs were found on the Mouse Genome Informatics website. DNA was extracted from mouse tail tips with DNA Isolation Mini Kit (Vazyme). PCR Products were separated by 3% agarose gels and visualized by ethidium bromide staining. Primers were listed in Table S4 .
5
Quantification of Viral Genomic Copies
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Virus load was determined by viral genomic copies using qPCR. DNA was extracted using the DNA isolation Mini Kit (Vazyme, Nanjing, China). The levels of viral genomic copies were quantified by the amount of viral major capsid protein (mcp) gene copies, which were determined with the method of standard curve. Specific primers and probes were designed from the partial MRV mcp sequence of NH-1609 using Primer Express v3.0 program. The forward primer 5′-GCCAAAAACCTTGTCCTTCCT -3′, reverse primer 5′-TCGTTGTAAGGCAGGGTGACT-3′, and the probe 5ʹ-FAM-CCCTTCTTTTTCGGCAGAGA-BHQ1-3ʹ were identified. Each PCR mixture in 10 µL contained 5 µL 2× Real PCR Easy TM Mix-Taqman (FOREGENE, Chengdu, China), 400 nM primer each, 200 nM probe, and 100 ng total DNA template. The program started with an initial denaturation at 94 °C for 3 min, followed by 40 cycles at 94 °C for 10 s and 60 °C for 30 s. The amplification and date analysis were carried out in Roche LightCycler® 480 (Roche Diagnostics, Switzerland).
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