RNA-immunoprecipitation was performed in accordance with the standard protocols with minor modifications [32 (link), 33 (link)]. Embryos were cross-linked with 1% formaldehyde in PBS and incubated in ice-cold nuclear isolation buffer [0.32 M sucrose, 10 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 1% Triton X-100] for 20 min with frequent mixing. After centrifugation for 15 min at 2500g, the resulting nuclei pallet was incubated with RIP buffer [150 mM KCl, 25 mM Tris (pH 7.4), 5 mM EDTA, 0.5 mM DTT, 0.5% NP40, 120 U/ml RNase inhibitor, protease inhibitor cocktail]. Anti-Jmjd6 antibody (LS-C30343, LSbio) or rabbit IgG (12–370, Millipore) coupled to protein G agarose bead (SC-2002, Santa Cruz Biotechnology) was incubated with the nuclear extracts for overnight at 4°C. After washing the beads with ice-cold RIP buffer, DNase I (2270A, Takara) was treated. The RNAs were extracted with phenol/chloroform and precipitated by ammonium acetate/ethanol. Oligonucleotides are listed in Supporting Information (S1 Table ).
Dnase 1 2270a
Manufactured by Takara Bio
Sourced in United States, Japan
DNase I (2270A) is a laboratory product offered by Takara Bio. It functions as a deoxyribonuclease enzyme that cleaves DNA, enabling the removal of DNA from samples.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using dnase 1 2270a
1
RNA Immunoprecipitation Protocol with Modifications
2
Virucidal Pretreatment of Male Genital Fluids
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For the pre-treatment of viruses with fluids from the male genital tracts, we incubated viruses with the defined concentration of the fluids in complete medium at 37°C for the specific durations. For heat treatment, SP and PF were heated in boiling water for 15 min. After a centrifugation at 12,000 × g for 10 min, supernatants were collected. To treat samples with enzymes, SP and PF were incubated with individual enzymes, including 300 µg/ml proteinase K (ProK) (P6565, Sigma-Aldrich, St. Louis, MO, USA), 200 U/ml DNase I (2270A, Takara Bio., Dalian, China), 200 µg/ml RNase A (GE101, TransGen Biotech, Beijing China), 200 U/ml PNGase F (G5166, Sigma), and 100 U/ml lipase (L8620, Solarbio Life Sciences Ltd. Co, Beijing, China), at 37°C for 5 h. SP and PF were then heated in boiling water for 5 min to inactivate enzymes.
3
Quantification of HBV DNA and pgRNA
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HBV DNA in transfected cells culture supernatant was treated with PNE solution (8.45% polyethylene glycol (PEG), 0.445 mol NaCl, 13 mmol EDTA) in ice for 1 h, and incubated with Dnase I (2270A, TAKARA, Shiga, Japan) and Rnase (2158, TAKARA , Shiga, Japan) at 37 °C for 1 h. Microspheres were treated with proteinase K at 56 °C for 12 h, and HBV DNA was isolated by phenol/chloroform extraction-ethanol precipitation method. HBV DNA copy number was determined by quantitative polymerase chain reaction (qPCR). To quantify HBV 3.5 kb pgRNA, total RNA was extracted from HBV transfected cells using total RNA extractor (TRI) reagent (TR 118, Molecular Research Center, Cincinnati, OH, USA). After treatment, using Dnase I and Rnase inhibitor, cDNA template was synthesized. Unspliced 3.5 kb RNA was quantified by qPCR using Synergy BrandsSynergy Brands (SYBR) qPCR Mix kit (QKD-201T, Toyobo, Osaka, Japan). The thermal cycling conditions comprised 1 min at 95 °C, followed by 40 cycles at 95 °C for 15 sec and 60 °C for 30 sec. Primer sequences used in this study were: 5′-TCCCTCGCCTCGCAGACG-3′ and 5′-GTTTCCCACCTTATGAGTC-3′ for unspliced 3.5 kb RNA, and 5′-TTCTACAATGAGCTGCGTGTG-3′ and 5′-GGGGTGTTGAAGGTCTCAAA-3′ for β-actin mRNA.
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