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Oligonucleotides

Manufactured by Ajinomoto Group
Sourced in Japan

Oligonucleotides are short, synthetic DNA or RNA sequences used in various laboratory applications. They serve as building blocks for the synthesis and analysis of genetic material. Oligonucleotides can be utilized in a range of techniques, including polymerase chain reaction (PCR), DNA sequencing, and gene expression studies.

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3 protocols using oligonucleotides

1

Oligonucleotide Synthesis and Modifications

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All Oligonucleotides used in this study contained phosphorothioate linkages. Oligonucleotides (except for those modified with ENA) were synthesized and purified by GeneDesign (Osaka, Japan). ENA-modified Oligonucleotides were synthesized and purified by Sigma-Aldrich Japan (Tokyo, Japan). LNA with unmethylated cytosine was prepared according to the methods described in previous literature7 (link),25 (link),26 (link). Oligonucleotides with 2′-MCE and BNANC(N-Me) were kindly provided by GeneDesign. For 2′-MCE-modified Oligonucleotides, 2′-MCE-modified uridines were used in the positions of sugar-modified thymidines in other sugar-modified Oligonucleotides. Oligonucleotides were suspended in sterile, endotoxin-free water (Thermo Fisher Scientific) and subjected to the TLR9 activation assay.
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2

Synthesis of Radiolabeled dsRNA Substrates

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Radiolabeled dsRNA was prepared as described previously (Iki et al. 2017 ). Briefly, 34/36-nt dsRNA was prepared from two oligonucleotides (Gene Design). The 34-nt strand was end-labeled by incubation with [γ-32P]-dATP and T4 PNK (Thermo Scientific), whereas 36-nt strand was end-phosphorylated without radiolabeling. Single-stranded RNAs were annealed as follows: Guide and passenger strand were mixed together with 5× annealing buffer (Tris–HCl [pH 7.6] 50 mM, KCl 100 mM, and MgCl2 5 mM) and incubated for 2 min at 96°C in a thermomixer (Eppendorf), at which point the machine was turned off and ssRNAs were left to anneal with decreasing temperature for 12 h. 98/100- and 510/512-nt dsRNA substrates were created by in vitro transcription of individual strands using the SP6-Scribe Standard RNA IVT kit (Cellscript) with [α-32P]-CTP, purified on mini Quick Spin RNA Columns (Roche) and extracted with phenol:chloroform:isoamyl alcohol (PCI), followed by ethanol precipitation. Annealing of single-stranded RNA to obtain 98/100- and 510/512-nt dsRNA was achieved as described for 34/36-nt dsRNA species. All three species of duplexes contain a 5′ blunt end and 2 nt 3′ overhang.
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3

Mammalian Cell Expression Vectors and Reagents

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The expression vectors in mammalian cells, pAcGFP1-Actin, pAcGFP1-Tubulin, and pDsRed-Monomer-C1, were obtained from Clontech. Oligonucleotides used as PCR primers in the construction of expression plasmids for fusion proteins were custom-synthesized by Gene Design (Osaka, Japan). The WST-1 cell proliferation assay system was purchased from Takara Bio (Kyoto, Japan). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA). Glass-bottom dishes used for cell culture and imaging experiments were purchased from AGC Techno Glass (Shizuoka, Japan).
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