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35 protocols using nucleotide removal kit

1

5hmC Enrichment and Detection Protocol

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Genomic DNA was sonicated to ~200 bp by Bioruptor (each cycle with 30 s on and 30 s off, 13 cycles in total). The 5hmC enrichment was done, as previously described, with an improved selective chemical labeling method. The 5hmC labeling reactions were performed in a 30-μl solution containing 50 mM HEPES buffer (pH 7.9), 25 mM of MgCl2, 5–10 ug of sonicated genomic DNA, 100 μM of UDP-6-N3-Glu (Jena Bioscience), and 15 units of wild-type β-glucosyltransferase (New England Biolabs). Reactions were incubated for 1 h at 37°C. DNA substrates were purified via a Nucleotide removal kit (Qiagen) and reconstituted in H2O. About 4 ul of 1 mM DBCO-S-S-peg3-biotin was added into the DNA solution for incubation for 2 h at 37°C. Then, samples were purified by a DNA purification kit (Qiagen) following the manufacturer's recommendations. The biotin-labeled DNA was enriched by Streptavidin-coupled Dynabeads (Dynabeads® MyOneTM Streptavidin T1, Life Technologies) and purified by a Qiagen Minelute purification kit.
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2

Targeted Biotinylated DNA Sequencing

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Custom long oligonucleotide ‘ultramer’ probes (33-plex, Panel A) were obtained from IDT (Integrated DNA Technologies). NEBNext Direct™ Cancer hotspot panel (Panel B) and xGen Pan Cancer™ panel (Panel C) were purchased from NEB and IDT, respectively.
TdT (from NEB) reaction was performed on a thermocycler (Mastercycler Nexus, Eppendorf) to generate poly-adenines at the 3′end of ssDNA prior to BST–DSN reaction. The protocol is described in Supplementary Table S3. The products generated from TdT reaction were then employed in a 10 μl final volume of BST–DSN reaction master mix containing Biotin-11-dUTP (B-dUTP) and anchored-oligo-dT (Supplementary Table S3). Nucleotide removal kit (Qiagen) was used to purify the TBD products and the size of TBD products was analyzed on an Agilent DNA Chip 1000 analyzer (Agilent). Reactions were repeated at least three times to check the repeatability of results.
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3

Quantitative SAN1 Enzyme Kinetics

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Reactions were carried out with varied concentrations of substrate (10–1000 nM) and 240 pmol enzyme in Elution Buffer. Reactions containing SAN1 WT and substrate were pre-warmed to 37 °C and initiated by the addition of Start Buffer. Reactions were sampled at three time points by addition of Stop Buffer, and processed using the Nucleotide Removal kit (Qiagen). Products were quantified using Ecoscint Original scintillation liquid (National Diagnostics) and a scintillation counter and were analyzed using GraphPad Prism.
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4

BAC Clone DNA Isolation and Labeling

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BAC clone DNA was isolated using the Qiagen miniprep kit prior to amplification and direct labeling by nick translation. Probes were labeled with Texas red-12-dUTP (Invitrogen) and FITC-fluorescein-12-UTP (Roche) prior to purification using the Qiagen nucleotide removal kit.
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5

Electrochemical Analysis of Nucleic Acids

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Nucleosides, dNTPs and other building blocks were analyzed using conventional in situ cyclic voltammetry (CV). The PEX products were analyzed using ex situ (adsorptive transfer stripping, AdTS) CV or square-wave voltammetry (SWV). The PEX products (purified in their single-stranded form using streptavidin-coated magnetic beads or in their double-stranded forms using a Qiagen Nucleotide Removal Kit) were accumulated at the surface of a working electrode (hanging mercury drop electrode, HMDE) for 60 s, from 5 μL aliquots containing 0.2 M NaCl. The electrode was then rinsed with deionized water and placed into a electrochemical cell. CV settings: scan rate 1 V s–1, initial potential 0.0 V, for switching potentials see figure legends. SWV settings: initial potential 0 V, for final potentials see figure legends; frequency 200 Hz, amplitude 50 mV. Background electrolyte: 0.5 M ammonium formate, 0.05 M sodium phosphate, pH 6.9. All measurements were performed at room temperature using an Autolab analyzer (Eco Chemie, The Netherlands) in connection with VA-stand 663 (Metrohm, Herisau, Switzerland). The three-electrode system was used with a Ag/AgCl/3 M KCl electrode as a reference and platinum wire as an auxiliary electrode. Measurements of reduction signals were performed after deaeration of the solution by argon purging.
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6

DNA Gap Filling and Labeling Protocol

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40 pmol DNA was incubated with 3 μg wild-type TDG, 13.3 fg APE1 D308A, and 4 µg bovine serum albumin (BSA, New England Biolabs, Ipswitch, MA) in 20 µL HEMN.1 Buffer (200 mM HEPES, 1 M NaCl, 2 mM EDTA, 25 mM MgCl2) at 37 °C for 1 h to excise target bases and detach the TDG from the resulting AP site. After purifying the DNA with a Nucleotide Removal Kit (Qiagen, Valencia, CA), it was incubated with 20 U of Endonuclease IV (New England Biolabs), 100 U of Endonuclease VIII (New England Biolabs), and 4 µg BSA in 20 µL NEB2 buffer (50 mM NaCl, 10 mM tris–HCl, 10 mM MgCl2, 1 mM DTT, New England Biolabs) at 37 °C for 30 min to prime the gap for base incorporation. Then, 1.5 nmol of biotin-11-dCTP (C28H44N7O16P3S, Perkin Elmer, Waltham, MA) and 0.12 U of T4 polymerase having no exonuclease activity (Lucigen, Middleton, WI) were added and the mixture and incubated at 37 °C for an additional 30 min. The DNA was again purified with the Nucleotide Removal Kit and eluted in deionized water.
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7

Fluorescent Probe Labeling and Purification

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Secondary “readout” probes were purchased from Thermo Fisher with 5’ C5 amine and 3’ C7 amine modifications. 5 ug of each readout probe was then coupled to the appropriate fluorescent dye according to the manufacturer’s specifications. After labeling, probes were extracted from excess dye by use of a Qiagen Nucleotide Removal Kit, resuspended in Ultrapure water, and stored at −20°C. Labeling efficiency was determined using Beer’s law. Only fluorescently labeled probes with >1.5 dyes/oligonucleotide were utilized in these studies.
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8

Avian Microchromosome BAC Probes

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Two BACs, selected from chicken galgal4 assembly (Hillier et al. 2004 (link)) and positioned as close as possible to each end of the chromosome, were chosen for each available reference microchromosome (GGA10-28 with the exception of GGA16) from the universal avian zooFISH probe set developed in our previous study by Damas et al. (2017 (link)) (Table S1). BAC clone DNA was isolated using the Qiagen miniprep kit prior to amplification and direct labelling by nick translation. Probes were labeled with Texas red-12-dUTP (Invitrogen) and FITC-fluorescein-12-UTP (Roche) prior to purification with the Qiagen nucleotide removal kit.
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9

Synthetic Guide RNA Production

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55-base oligomers encoded with T7 promoter sequence (5′-TTCTAATACGACTCACTATAG-3′), 20-base target sequence, and an overlap sequence (5′-GTTTTAGAGCTAGA-3′) were designed and purchased from IDT. An 80 base long overlap-complementary sequence (5′-AAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3′) was also purchased from IDT. Briefly, 10 µM of equimolar pooled oligomers and 10 µM of overlap-complementary overlap sequence containing oligomer are first mixed, denatured at 95 °C for 15 s, and later allowed to hybridize at 43 °C for 5 min in 1× NEBuffer 2.0 (NEB). The hybridized oligos were then extended with 5U of Klenow exo- at 37 °C for 1 h in the presence of 2 mM dNTPs. Next, an exonuclease treatment was carried out at 37 °C for 1 h with 10U of Exonuclease I (NEB) in 1× Exonuclease buffer (NEB). The dsDNA was purified with a Qiagen Nucleotide removal kit and quantified via absorbance spectroscopy before use in RNA synthesis. Using the above dsDNA, a single guide RNA mixture (sgRNA) was synthesized following the manufacturer’s recommendations (HiScribe T7 High Yield RNA Synthesis Kit, NEB). After transcription and DNaseI (NEB) treatment, the sgRNA was purified using spin columns (Monarch RNA Cleanup Kit T2030, NEB) and quantified via absorbance spectroscopy before use. The 260/280 ratio for dsDNA was ~ 1.8–1.9 and the synthesized sgRNA was ~ 2.0.
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10

Tyrosine-Linked DNA Substrate Assay

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All single-standard DNA substrates share the commons sequence of 5′-TCCGTTGAAGCCTGCTTT-3′. Oligonucleotides were synthesized in the reversed direction, as described previously (42 (link)). CBV was prepared according to the literature (43 (link)). Total cell lysates from DT40 Tdp1−/− and Tdp1−/−hTDP1 cells were prepared in the same manner as previously described (44 (link)). The protein concentration of lysates was determined by Bradford protein assay (Quick Start protein assay, Bio-Rad Laboratories). To prepare DNA substrate, oligonucleotides with 3-phosphotyrosine and CBV linkages synthesized were incubated with [γ-32P]adenosine triphosphate (NEG502A, PerkinElmer Life Sciences) and T4 polynucleotide kinase (Takara) for 5′-end labeling. After being purified using a nucleotide removal kit (Qiagen), 1 nM labeled oligonucleotide was incubated with cell lysates at 25°C for 15 min. Samples were separated by 16% denaturing polyacrylamide gel electrophoresis (7 M, urea). Dried gel was exposed on the imaging plate (Fujifilm) and then scanned in Fuji Bas 2500 system. Quantification was performed by Image Gauge software (Fujifilm).
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