Dntps

Manufactured by Illumina

DNTPs (Deoxyribonucleotide Triphosphates) are essential chemical building blocks used in various DNA-based applications. They serve as substrates for DNA synthesis, enabling the replication and amplification of genetic material.

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Lab products found in correlation

Rnaseh, by Illumina (2 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions) Dna polymerase 1, by Illumina (2 mentions) Truseq rna sample prep kit, by Illumina (1 mentions) Magnetic beads, by Illumina (1 mentions) Hiseq 2500 platform, by Illumina (1 mentions) Random hexamer primers, by Illumina (1 mentions) Poly t oligo attached magnetic beads, by Illumina (1 mentions) Nucleospin rna kit, by Takara Bio (1 mentions)

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DNTP mixture (2 citations)

2 protocols using dntps

RNA-Seq Library Construction and Sequencing

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Construction of the four libraries and the RNA-Seq were performed by the Biomarker Biotechnology Corporation (Beijing, China). Five micrograms of total RNA from each sample (EM, EH, LM, and LH) were used to construct the sequencing libraries. RNA sequencing libraries were generated using the TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA) following the manufacturer’s recommendations. Briefly, the poly (A)-containing mRNA molecules were purified from the total RNA by using the poly-T oligo-attached magnetic beads (Illumina, San Diego, CA, USA). Then, mRNA was broken into short fragments, which were used as templates for cDNA synthesis. Double-stranded cDNA was synthesized using SuperScript II, buffer, dNTPs, RNaseH, DNA polymerase I, and random hexamer primers (Illumina). After that, the ‘A’ tail was added to the 3′ ends of the repaired cDNA fragments and the Illumina’s paired-end adapters were ligated to the cDNA ends. The products from the ligation reaction were amplified by PCR. The PCR productions were purified by magnetic beads (Illumina) and dissolved in EB solution to generate the sequencing libraries. The quantity and quality of each sequencing library were tested by the Agilent 2100 Bioanalyzer. Finally, the four libraries were sequenced separately on Illumina HiSeq™ 2500 platform.

Li W., Zhang L., Ding Z., Wang G., Zhang Y., Gong H., Chang T, & Zhang Y. (2017). De novo sequencing and comparative transcriptome analysis of the male and hermaphroditic flowers provide insights into the regulation of flower formation in andromonoecious taihangia rupestris. BMC Plant Biology, 17, 54.

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RNA-seq Protocol for Gene Expression Analysis

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RNA was purified by using NucleoSpin RNA kit ((Takara Bio USA, Inc Cat #740955) according to the manufacturer’s instruction. RNA quality was validated by RNA integrity number (RIN >9) calculated by Agilent 2100 Bioanalyzer. mRNA was enriched using oligo(dT) beads and then was fragmented randomly by adding fragmentation buffer. The cDNA was synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina, dNTPs, RNase H and DNA polymerase I) was added to initiate the second-strand synthesis. After a series of terminal repair, a ligation and sequencing adaptor ligation, the double-stranded cDNA library is completed through size selection and PCR enrichment. The qualified library was fed into Illumina sequencer after pooling and more than 30 million reads was acquired for each sample. The quality of RNA-seq raw reads were checked using FastQC (Galaxy Version 0.72). Raw reads were mapped to human genome, hg19, using RNA STAR. Differential expression and gene set enrichment analysis was assessed using edgeR 3.24.3 and limma 3.38.3 with R 3.5. Computational analysis was performed using Galaxy server (https.usegalaxy.org). The RNAseq data was submitted to the Gene Expression Omnibus (GEO) repository at the National Center for Biotechnology Information (NCBI) archives with assigned GEO accession numbers of GSE162487.

Guan Y., Tiwari A.D., Phillips J.G., Hasipek M., Grabowski D.R., Pagliuca S., Gopal P., Kerr C.M., Adema V., Radivoyevitch T., Parker Y., Lindner D.J., Meggendorfer M., Abazeed M., Sekeres M.A., Mian O.Y., Haferlach T., Maciejewski J.P, & Jha B.K. (2020). A Therapeutic Strategy for Preferential Targeting of TET2-Mutant and TET Dioxygenase–Deficient Cells in Myeloid Neoplasms. Blood Cancer Discovery, 2(2), 146-161.

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