Plugs were treated with sequential combination of Exonuclease VII (NEB) for 1 hr at 37°C followed by Exonuclease T (NEB) for 45 min at 24°C to blunt DNA ends before Illumina adapter ligation (Canela et al., 2019 (link)). Subsequent steps of A-tailing, adapter ligation, plug melting, chromatin shearing, and second round of adapter ligation for sequencing were performed exactly as previously described (Canela et al., 2019 (link); Canela et al., 2017 (link)).
Exonuclease t
Manufactured by New England Biolabs
Sourced in China
Exonuclease T is a DNA-specific exonuclease that catalyzes the stepwise removal of mononucleotides from the 3' end of double-stranded or single-stranded DNA. It has no activity on RNA.
Automatically generated - may contain errors
Lab products found in correlation
Ribolock rnase inhibitor, by Thermo Fisher Scientific (3 mentions)
T4 polynucleotide kinase, by Thermo Fisher Scientific (2 mentions)
Rt pcr grade water, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Direct zol rna kit, by Zymo Research (2 mentions)
Rna clean and concentrator kit, by Zymo Research (2 mentions)
Nanodrop, by Agilent Technologies (2 mentions)
Exonuclease 7, by New England Biolabs (1 mentions)
Sybr gold, by Thermo Fisher Scientific (1 mentions)
Oligodnas, by Thermo Fisher Scientific (1 mentions)
T4 rna ligase 2 rnl2, by New England Biolabs (1 mentions)
Lactate dehydrogenase, by Merck Group (1 mentions)
Pyruvate kinase, by Merck Group (1 mentions)
S adenosyl methionine sam, by Merck Group (1 mentions)
Rneasy rna purification kit, by Qiagen (1 mentions)
Atpγs, by Merck Group (1 mentions)
Adpnp, by Merck Group (1 mentions)
Adenosine triphosphate (atp), by Thermo Fisher Scientific (1 mentions)
Novex 15 tbe urea gel, by Thermo Fisher Scientific (1 mentions)
Sybr gold nucleic acid gel stain, by Thermo Fisher Scientific (1 mentions)
8 protocols using exonuclease t
1
Blunt DNA Ends for Illumina Sequencing
2
RNA and DNA Ligation Protocol
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All oligoRNAs used in this study were synthesized by GenScript (Nanjing, China), and the oligoDNAs were purchased from Invitrogen (Shanghai, China). For ligation experiments, a phosphate was enzymatically introduced to the 5′-position of RNA or DNA by using T4 polynucleotide kinase (Thermo Scientific; Pittsburgh, PA, USA). SYBR Gold and RiboLock RNase Inhibitor were also from Thermo Scientific. T4 RNA ligase 2 (Rnl2) and Exonuclease T were purchased from NEW ENGLAND BioLabs (Beijing, China). All other chemicals were from Sigma-Aldrich (St. Louis, MO, USA).
3
Protocol for Nucleotide Preparation and Assays
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ATP, GTP, CTP, and UTP (products 10585, 16800, 14121, 23160, respectively) were purchased from Affymetrix (Santa Clara, CA). [α-32P]-GTP was from PerkinElmer (product BLU006H250UC) (Waltham, MA). ATP-γ-S, ADPCP, and ADPNP (products A1388, M7510, and A2647, respectively), S-adenosyl methionine (SAM) (product A7007), and the pyruvate kinase (900–1400 units/mL)/lactate dehydrogenase (600–1000 units/mL) mix from rabbit muscle (product P0294) were from Sigma (St. Louis, MO). NADH disodium salt was from Calbiochem (product 481913) (San Diego, CA). Phosphoenolpyruvate potassium salt was purchased from Chem Impex International, Inc. (product 09711) (Wood Dale, IL). RiboLock RNase inhibitor was from Thermo Fisher Scientific (product EO0381) (Waltham, MA). The RNeasy RNA purification kit was purchased from Qiagen (product 74106) (Germany). Exonuclease T was purchased from New England Biolabs (product M0265S) (Ipswich, MA). The Abnova Small RNA Marker was purchased from Abnova (product number R0007) (Taiwan). SYBR Gold nucleic acid gel stain (product S11494) and Novex 15% TBE-Urea gels (product EC68852BOX) were purchased from Thermo Fisher Scientific. Corning 384-well plates were purchased from VWR International (product 3544) (Radnor, PA).
4
RNA Ligation Protocol for Researchers
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All oligoRNAs used in this study were synthesized by GENEWIZ, Inc. (Suzhou, China), and the sequences are listed in Supplementary Table S1 . For ligation experiments, a phosphate was in advance introduced to the 5′-position of L-RNA by using T4 polynucleotide kinase (Thermo Scientific; Pittsburgh, PA, USA). RiboLock RNase Inhibitor was also from Thermo Scientific. Ultra GelRed was from Vazyme (Nanjing, China). T4 Rnl2 and Exonuclease T were purchased from NEW ENGLAND BioLabs (Beijing, China). All other chemicals were from Sigma-Aldrich (St. Louis, MO, USA).
5
Ribonuclease A Enzymatic Assay
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Ribonuclease A was obtained from Sigma, and RNase T1 (100 000 U/ml) was from Roche. Exonuclease T and Murine RNase inhibitor (40 U/μl) were from New England Biolabs. N-Methylmesoporphyrin IX (NMM) was obtained from Frontier Scientific. Sterile Phosphate buffered saline, PBS (purchased as 10X DPBS), DMEM, trypsin–EDTA solution, fetal bovine serum and nuclease-free distilled water were obtained from Gibco.
6
RNA Extraction and Aminoacylation Characterization
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Cells were harvested using trypsin and washed with cold phosphate-buffered saline. Total RNA was extracted by Trizol (Invitrogen) and Direct-zol RNA kit (Zymo Research). RNA was kept in buffers at pH 5 at all times. For the aminoacylation characterizing assay, total RNA was separated into two fractions. One fraction was incubated on ice in 250 μl of 0.2 M sodium acetate with 4 μl acidic anhydride for 2 hours for N-acetylation as described previously [48 (link)]. The other fraction was incubated at 37 degrees at pH 9.0 for 30 min to deacylated the aminoacyl group [48 (link)]. Both fractions were then ethanol precipitated at -80 degrees for 2 hours and washed with 80% ethanol. 10 ug total RNA was resuspended in 87 μl RT-PCR grade water (ThermoFisher), and 3 μl exonuclease T (New England Biolabs, NEB) with 10 μl buffer 4 (NEB) was used in the enzymatic reaction. The digestion reactions were kept at room temperature for 1 hour, and then the RNA clean and concentrator kit (Zymo) was used to remove the exonuclease T and other residues. The RNA samples were quantified by nanodrop (Agilent) and subjected to subsequent applications.
7
Enzyme-Based DNA Modification Protocol
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The oligonucleotide sequences used in this work (Table S1 ) and nuclease-free water were purchased from Integrated DNA Technologies (Coralville, IA, USA). Moreover, 10× NEBuffer 1, exonuclease I, exonuclease III, exonuclease T, lambda exonuclease, Nt. AlwI, and T4 polynucleotide kinase were all purchased from New England Biolabs (Ipswich, MA, USA). Nanoceria dispersion (catalog number 289744, 20 wt% dispersed in 2.5% acetic acid), 3,3′,5,5′-tetramethylbenzidine (TMB, catalog number T0440), human serum (catalog number H4522), 1 M HEPES buffer solution (pH 5.0) (Figure S7 ), sodium phosphate dibasic (Figure S7 ), sodium phosphate monobasic (Figure S7 ), acetic acid (Figure S7 ), and sodium acetate (Figure S7 ) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Finally, 1 M Tris-HCl buffer (pH 7.4) (Figure S7 ) was purchased from Bioneer (Daejeon, Republic of Korea).
8
Aminoacylation Characterization Assay
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Cells were harvested using trypsin and washed with cold phosphate-buffered saline. Total RNA was extracted by Trizol (Invitrogen) and Direct-zol RNA kit (Zymo Research). RNA was kept in buffers of pH 5 at all time and avoid too many freeze-thaw cycles to keep the aminoacyl group attached to tRNA.
For aminoacylation characterizing assay, total RNA was separated into two fractions. One fraction was incubated on ice in 250 µl of 0.2 M sodium acetate with 4 µl acidic anhydride for 2 hours for Nacetylation as described previously (Walker & Fredrick, 2008) . The other fraction was incubated at 37 degrees at pH 9.0 for 30 min to deacylated the aminoacyl group (Walker & Fredrick, 2008) . Both fractions were then ethanol precipitated at -80 degrees for 2 hours and washed with 80% ethanol. 10 ug total RNA was resuspended in 87 µl RT-PCR grade water (ThermoFisher), and 3 µl exonuclease T with 10 µl buffer 4 (NEB) was used in the enzymatic reaction. The digestion reactions were kept at room temperature for 1 hour, and then the RNA clean and concentrator kit (Zymo) was used to remove the exonuclease T and other residues. The RNA samples were quantified by nanodrop (Agilent) and subjected to subsequent applications.
For aminoacylation characterizing assay, total RNA was separated into two fractions. One fraction was incubated on ice in 250 µl of 0.2 M sodium acetate with 4 µl acidic anhydride for 2 hours for Nacetylation as described previously (Walker & Fredrick, 2008) . The other fraction was incubated at 37 degrees at pH 9.0 for 30 min to deacylated the aminoacyl group (Walker & Fredrick, 2008) . Both fractions were then ethanol precipitated at -80 degrees for 2 hours and washed with 80% ethanol. 10 ug total RNA was resuspended in 87 µl RT-PCR grade water (ThermoFisher), and 3 µl exonuclease T with 10 µl buffer 4 (NEB) was used in the enzymatic reaction. The digestion reactions were kept at room temperature for 1 hour, and then the RNA clean and concentrator kit (Zymo) was used to remove the exonuclease T and other residues. The RNA samples were quantified by nanodrop (Agilent) and subjected to subsequent applications.
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