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5 protocols using hind 3

1

Quantitative Analysis of DNA Composition

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Cells were resuspended in TE buffer containing 62.5 μg/mL proteinase K (Invitrogen, AM2546), 62.5 μg/mL RNase A (Thermo Scientific, EN0531), and 0.5% SDS and incubated overnight at 37°C. Genomic DNA was purified by phenol/chloroform extraction and resuspended in RNase/DNase free water. 5 μg DNA was digested with 5 μL RNase H (NEB, M0297), 3 μL Hind III (Fisher, FD0504), 3 μL EcoRI (Fisher, FD0274), and 3 μL Bam HI (Fisher, FD0054) in RNase H buffer (NEB, M0297) overnight at 37°C. Digested DNA was purified using the GeneJET PCR Purification Kit (Thermo Scientific, K0702). DNA was further digested into single nucleosides using DNA Degradase Plus (Zymo Research, E2021). To quantitate DNA constituent base composition, a fit-for-purpose LC-MS/MS assay was implemented on a 1290 Infinity II Autosampler and Binary Pump (Agilent) and a SCIEX 6500+ triple quadrupole mass spectrometer (SCIEX). Chromatographic separation was conducted on an Inertsil ODS-3 (3 μm × 100 mm 2.1 mm) reverse phase column (GL Sciences) at ambient temperature with a gradient mobile phase of methanol and water with 0.1% formic acid. MRM transitions of all analytes and isotopic internal standards were monitored to construct calibration curves. We were able to quantitate 1 rN per 20,000 bases from 1 mg DNA.
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2

AFLP Analysis Protocol with Modifications

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The AFLP analysis was performed as described previously (Vos et al., 1995 (link)), with minor modifications according to Klie et al. (2013) (link). For each sample, 100 ng of DNA was digested with 9 U HindIII (Fisher Scientific – Germany GmbH, Schwerte, D) and 3.5 U MseI (Fisher Scientific - Germany GmbH, Schwerte, D). The preamplification reactions were performed with specific primers that had an A as a selective base at the 3′ end [HindIII (5′-AGACTGCGTACCAGCTT-A-3′) and MseI (5′-GACGATGAGTCCTGAGTAA-A-3′)]. HindIII (5′-AGACTGCGTACCAGCTT-ANN-3′) primers with two extra selective bases and MseI (5′-GACGATGAGTCCTGAGTAA-ANNN-3′) primers with three extra selective bases were used for the final amplification. The HindIII primers were end-labeled with an infrared dye (either IRD 700 or IRD 800; Eurofins MWG, Ebersberg, D). In a single PCR reaction, labeled primers were used either as single primers or in combinations of two differently labeled primers (IRD 700 and IRD 800). In total, 21 selective primer combinations were analyzed (Table 1). The fragments were separated on 6 % polyacrylamide gels (Sequagel XR, Hessle, UK) using a DNA analyzer (LI-COR, Lincoln, Nebraska, USA) and automatically processed using the e-Seq-Software (V3.0, LI-COR, Lincoln, Nebraska, USA).
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3

Quantitative Analysis of DNA Composition

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Cells were resuspended in TE buffer containing 62.5 μg/mL proteinase K (Invitrogen, AM2546), 62.5 μg/mL RNase A (Thermo Scientific, EN0531), and 0.5% SDS and incubated overnight at 37°C. Genomic DNA was purified by phenol/chloroform extraction and resuspended in RNase/DNase free water. 5 μg DNA was digested with 5 μL RNase H (NEB, M0297), 3 μL Hind III (Fisher, FD0504), 3 μL EcoRI (Fisher, FD0274), and 3 μL Bam HI (Fisher, FD0054) in RNase H buffer (NEB, M0297) overnight at 37°C. Digested DNA was purified using the GeneJET PCR Purification Kit (Thermo Scientific, K0702). DNA was further digested into single nucleosides using DNA Degradase Plus (Zymo Research, E2021). To quantitate DNA constituent base composition, a fit-for-purpose LC-MS/MS assay was implemented on a 1290 Infinity II Autosampler and Binary Pump (Agilent) and a SCIEX 6500+ triple quadrupole mass spectrometer (SCIEX). Chromatographic separation was conducted on an Inertsil ODS-3 (3 μm × 100 mm 2.1 mm) reverse phase column (GL Sciences) at ambient temperature with a gradient mobile phase of methanol and water with 0.1% formic acid. MRM transitions of all analytes and isotopic internal standards were monitored to construct calibration curves. We were able to quantitate 1 rN per 20,000 bases from 1 mg DNA.
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4

Chromatin Immunoprecipitation and DNA Detection

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Genomic DNA was prepared as above. 5 μg DNA was digested with 3 μL Hind III (Fisher, FD0504), 3 μL EcoRI (Fisher, FD0274), and 3 μL Bam HI (Fisher, FD0054) +/−5 μL RNase H (NEB, M0297) in RNase H buffer (NEB, M0297) overnight at 37°C. Digested DNA was incubated with 50 μL of protein A/G agarose beads (Santa Cruz, sc-2003) and 10 ul mouse serum (MP Biomedicals, 152282) in 1.5 mL PBS/0.5% Triton X100 for 2 h at 4°C. Digested DNA was then incubated with 50 μL of protein A/G agarose beads and 5 μL anti-S9.6 (Millipore, MABE1095) overnight at 4°C. Agarose beads were washed 3× for 3 min in PBS/0.5% Triton X100 binding buffer followed by PBS wash for 3 min. The beads were then incubated with 3 μL proteinase K for 2 h at 37°C. Nucleic acids were purified by phenol/chloroform extraction and ethanol precipitation. DNA was denatured in 50 μL TE and mixed with 50 μL of 0.8 M NaOH/20 mM EDTA solution for 10 min at 95°C then placed on ice. The solution was neutralized using sodium acetate pH 7.0 and DNA sample was transferred to Whatman Hybond N+ Blotting Membrane (Millipore Sigma, Z761079). Nucleic acids were cross-linked by to the membrane by baking at 60°C for 30 min followed by UV exposure. The membrane was blocked for 1 h with 5% milk in TBST and blotted for anti-ssDNA (1:1000, Millipore, MAB3034).
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5

Chromatin Immunoprecipitation and DNA Detection

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Genomic DNA was prepared as above. 5 μg DNA was digested with 3 μL Hind III (Fisher, FD0504), 3 μL EcoRI (Fisher, FD0274), and 3 μL Bam HI (Fisher, FD0054) +/−5 μL RNase H (NEB, M0297) in RNase H buffer (NEB, M0297) overnight at 37°C. Digested DNA was incubated with 50 μL of protein A/G agarose beads (Santa Cruz, sc-2003) and 10 ul mouse serum (MP Biomedicals, 152282) in 1.5 mL PBS/0.5% Triton X100 for 2 h at 4°C. Digested DNA was then incubated with 50 μL of protein A/G agarose beads and 5 μL anti-S9.6 (Millipore, MABE1095) overnight at 4°C. Agarose beads were washed 3× for 3 min in PBS/0.5% Triton X100 binding buffer followed by PBS wash for 3 min. The beads were then incubated with 3 μL proteinase K for 2 h at 37°C. Nucleic acids were purified by phenol/chloroform extraction and ethanol precipitation. DNA was denatured in 50 μL TE and mixed with 50 μL of 0.8 M NaOH/20 mM EDTA solution for 10 min at 95°C then placed on ice. The solution was neutralized using sodium acetate pH 7.0 and DNA sample was transferred to Whatman Hybond N+ Blotting Membrane (Millipore Sigma, Z761079). Nucleic acids were cross-linked by to the membrane by baking at 60°C for 30 min followed by UV exposure. The membrane was blocked for 1 h with 5% milk in TBST and blotted for anti-ssDNA (1:1000, Millipore, MAB3034).
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