Nucleotide removal kit

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.

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.

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.

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.

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.