Genomic DNA was isolated from vegetative Oxytricha cells using the Nucleospin Tissue Kit (Takara Bio USA, Inc.). DNA was sheared into 150bp fragments using a Covaris LE220 ultra-sonicator (Covaris). Samples were gel-purified on a 2% agarose-TAE gel, blunted with DNA polymerase I (New England Biolabs), and purified using MinElute spin columns (QIAGEN). The fragmented DNA was dA-tailed using Klenow Fragment (3′ -> 5′ exo-) (New England Biolabs) and ligated to Illumina adaptors following manufacturer’s instructions. Subsequently, 2.2μg of adaptor-ligated DNA containing 6mA was immunoprecipitated using an anti-N6-methyladenosine antibody (Cedarlane Labs) conjugated to Dynabeads Protein A (Invitrogen). The anti-6mA antibody is commonly used for RNA applications, but has also been demonstrated to recognize 6mA in DNA (Fioravanti et al., 2013 (link); Xiao and Moore, 2011 ). The immunoprecipitated and input libraries were treated with proteinase K, extracted with phenol:chloroform, and ethanol precipitated. Finally, they were PCR-amplified using Phusion Hot Start polymerase (New England Biolabs) and used for Illumina sequencing.
Phusion hot start polymerase
Manufactured by New England Biolabs
Sourced in United Kingdom
Phusion Hot Start polymerase is a high-fidelity DNA polymerase designed for accurate DNA amplification. It exhibits enhanced specificity and robustness, reducing non-specific amplification and providing reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Rnasin rnase inhibitor, by Promega (6 mentions)
Milli q filtered water, by Merck Group (5 mentions)
Amicon ultra spin concentrator, by Merck Group (5 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (1 mentions)
Klenow fragment 3 5 exo, by New England Biolabs (1 mentions)
Nucleospin tissue kit, by Takara Bio (1 mentions)
Minelute spin column, by Qiagen (1 mentions)
Le220 ultrasonicator, by Covaris (1 mentions)
Dna polymerase 1, by New England Biolabs (1 mentions)
Amicon spin concentrator, by Merck Group (1 mentions)
Gblock, by Integrated DNA Technologies (1 mentions)
Hf buffer, by New England Biolabs (1 mentions)
Strataclone pcr cloning kit, by Agilent Technologies (1 mentions)
Milli q, by Merck Group (1 mentions)
T7 rna polymerase, by Promega (1 mentions)
8 protocols using phusion hot start polymerase
1
6mA Methylation Profiling in Oxytricha
2
Transcription Protocol for RNA Production
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DNA templates for transcription were ordered as gBlocks from Integrated DNA technologies (IDT) and cloned into pUC19 and sequenced. DNA was amplified for transcription by PCR using custom DNA primers and Phusion Hot Start polymerase (New England BioLabs). Transcription reactions were conducted using 500 μl PCR reactions as template into 5 ml transcriptions. Transcription reactions contained: ∼0.1 μM template DNA, 8 mM each NTP, 60 mM MgCl2, 30 mM Tris pH 8.0, 10 mM DTT, 0.1% spermidine, 0.1% Triton X-100 and T7 RNA polymerase as well as 5 μl RNasin RNase inhibitor (Promega). Tris was buffered at room temperature. Transcription reactions were left overnight, inorganic pyrophosphates were removed by centrifugation, and the reactions were ethanol precipitated and purified by denaturing PAGE purification. RNAs were eluted overnight at 4°C into ∼40 ml of diethylpyrocarbonate (DEPC)-treated milli-Q filtered water (Millipore) and concentrated using Amicon spin concentrators (Millipore).
3
In Vitro Transcription of RNA
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DNA templates for in vitro transcription were amplified by PCR using custom DNA primers (IDT) and Phusion Hot Start polymerase (New England BioLabs). Transcription reaction mixtures (2.5 ml) were assembled using 1,000-µl PCR reaction mixtures as the template (∼0.2 µM template DNA, a 6 mM concentration of each NTP, 60 mM MgCl2, 30 mM Tris [pH 8.0], 10 mM dithiothreitol [DTT], 0.1% spermidine, 0.1% Triton X-100, T7 RNA polymerase, and 2 µl RNasin RNase inhibitor [Promega]) and incubated overnight at 37°C. After inorganic pyrophosphates were precipitated by centrifugation, the reaction mixtures were ethanol precipitated and purified on a 7 M urea–8% denaturing polyacrylamide gel. RNAs of the correct size were excised, eluted overnight at 4°C into ∼40 ml of diethylpyrocarbonate (DEPC)-treated Milli-Q filtered water (Millipore), and concentrated using Amicon Ultra spin concentrators (Millipore). Mutations were introduced using mutagenized custom DNA reverse primers (Table 3 ).
4
In Vitro Transcription of RNA
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DNA templates for in vitro transcription were amplified by PCR using custom DNA primers (IDT) and Phusion Hot Start polymerase (New England BioLabs). A total of 2.5 mL transcription reactions was assembled using a 1000 µL PCR reaction, containing ∼0.2 µM template DNA. This template DNA was combined with 6 mM each NTP, 60 mM MgCl2, 30 mM Tris pH 8.0, 10 mM DT, 0.1% spermidine, 0.1% Triton X-100, T7 RNA polymerase, and 1 µL RNasin RNase inhibitor (Promega) and incubated overnight at 37°C. Next, inorganic pyrophosphates were removed through centrifugation. The reaction was ethanol precipitated and purified on a 7 M urea 8% denaturing polyacrylamide gel. Correct-sized RNA was cut from the gel and eluted in ∼50 mL of diethylpyrocarbonate (DEPC)-treated milli-Q filtered water (Millipore), washed, and concentrated using Amicon Ultra spin concentrators (Millipore).
5
Cd14 Promoter Truncation Analysis
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Promoter truncations were prepared by PCR using Phusion Hot Start Polymerase and 5xHF-Buffer as preset by the manufacturer (NEB, Ipswich, UK), utilizing primers and annealing temperatures depicted in Supporting Information (S1 Table ). Linearized pGL4.17 plasmid (Promega, Wisconsin, USA) containing Cd14 full-length promoters of C3Bir respectively B6 were served as templates [4 (link)]. For quality control the promoter fragments of C3Bir were flanked by KpnI and BglII,while B6 elements were flanked by XhoI and BglII restriction sites. Cloning of the truncated promoter elements was performed using StrataClone PCR Cloning Kit (Agilent, San Diego, CA) according to the suppliers recommendations. The promoter sequences were subsequently excised from pSC-A amp/kan with XhoI/BglII or KpnI/BglII (NEB) and inserted into XhoI/BglII or KpnI/BglII digested luciferase reporter plasmid pGL4.17.
6
In vitro Transcription of RNA
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DNA templates for in vitro transcription were amplified by PCR using custom DNA primers (IDT) and Phusion Hot Start polymerase (New England BioLabs). An amount of 2.5 mL transcription reactions were assembled using 1000 µL PCR reactions as template (∼0.2 µM template DNA), 6 mM each NTP, 60 mM MgCl2, 30 mM Tris pH 8.0, 10 mM DTT, 0.1% spermidine, 0.1% Triton X-100, T7 RNA polymerase, and 2 µL RNasin RNase inhibitor (Promega) and incubated overnight at 37°C. After inorganic pyrophosphates were precipitated by centrifugation, the reactions were ethanol precipitated and purified on a 7 M urea 8% denaturing polyacrylamide gel. RNAs of the correct size were excised, eluted overnight at 4°C into ∼40 mL of diethylpyrocarbonate (DEPC)-treated milli-Q filtered water (Millipore) and concentrated and washed using Amicon Ultra spin concentrators (Millipore).
7
RNA Purification and Synthesis Protocol
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Template DNA was amplified by PCR using custom DNA primers (Table S3 ) and recombinant Phusion Hot Start polymerase (New England Biolabs). In vitro transcription was carried out in a volume of 2.5 ml comprising 1.0 ml of PCR as the template. The transcription reaction mixture contained ∼0.2 M template DNA, a 6 mM concentration of each rNTP (ribonucleoside triphosphate), 60 mM MgCl2, 30 mM Tris (pH 8.0), 10 mM dithiothreitol (DTT), 0.1% spermidine, 0.1% Triton X-100, T7 RNA polymerase, and 2 μl RNasin RNase inhibitor (Promega). The transcription reaction mixture was incubated overnight at 37°C. The RNA was precipitated with 4 volumes of ice-cold 100% ethanol, incubating overnight at –20°C. Precipitated RNA was gel purified via the use of 7 M urea–8% denaturing polyacrylamide gel electrophoresis (dPAGE)–1× Tris-borate-EDTA (TBE). The RNA was visualized by UV light, excised from the gel, and eluted from the gel by the crush and soak method overnight at 4°C, using ∼50 ml of diethylpyrocarbonate (DEPC)-treated Milli-Q (Millipore) filtered water. Amicon Ultra spin concentrators (Millipore-Sigma) (30,000 molecular weight cutoff [MWCO]) were used to concentrate the eluted RNA to 2.5 mg/ml, and the reaction mixture was then stored at –20°C in DEPC-treated H2O until use.
8
High-yield RNA synthesis from DNA templates
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DNA templates for in vitro transcription were amplified by PCR using custom DNA primers (IDT) and Phusion Hot Start polymerase (New England BioLabs). 2.5 mL transcription reactions were assembled using 1000 µL PCR reactions as template (~0.2 µM template DNA), 6 mM each NTP, 60 mM MgCl2, 30 mM Tris pH 8.0, 10 mM DTT, 0.1% spermidine, 0.1% Triton X-100, T7 RNA polymerase and 2 µL RNasin RNase inhibitor (Promega) and incubated overnight at 37°C. After inorganic pyrophosphates were precipitated by centrifugation, the reactions were ethanol precipitated and purified on a 7 M urea 8% denaturing polyacrylamide gel. RNAs of the correct size were gel-excised, eluted overnight at 4°C into ~40 mL of diethylpyrocarbonate (DEPC)-treated milli-Q filtered water (Millipore) and concentrated and washed using Amicon Ultra spin concentrators (Millipore).
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