GSM8943852: Oxidized guanines, WT HAP1, DMSO, Biol Rep3; Homo sapiens; OTHER
In case of HAP1 and U2OS, cells were harvested by removing the medium, washing once with 10 mL DPBS followed by addition of 1 mL 0.25% Trypsin EDTA solution (Gibco). After an incubation of 5 min at 37°C, 9 mL medium was added, cells resuspended and transferred to a Falcon centrifugal tube. Cells were centrifuged (4°C, 0.3 rcf, 5 min) and the supernatant removed. gDNA was extracted from  cell pellets using the Monarch Genomic DNA Extraction Kit following the manufacturer's instructions. In order to avoid artefactual oxidation, DPBS used in the cell pellet resuspension  was supplemented with 1 mM deferoxamine and 50 µM n-tert-butyl-α-phenylnitrone. In case of the plasmid samples, the input plasmid vector pEGFP-W, derived from pEGFP-N1 (Addgene, Catalog Number 6085-1), was purified from BL21 (DE3) E. coli (NEB) using a Qiagen plasmid maxi prep kit (Qiagen, USA). To minimize artefactual damage, samples were inverted or flicked, instead of pipette mixing, until the CuAAC step. For DNA oxidation mapping, gDNA was eluted in H2O supplemented with 500 µM 8-hydroxyquinoline (HQ; Sigma) after extraction and purification. Undesired background modifications were blocked by adding 1 µL ENDOIV (NEB, 10 U/µL) and 500 µM 8-HQ to 5 µg of gDNA, freshly extracted from HAP1 pellets, in 1x ThermoPol Buffer (NEB) in a final volume of 20 µL, and incubating the mixture for 20 min at 37°C. 2.5 µL ddNTP mix (Jena Biosciences, 2.5 mM), 0.5 µL 10x ThermoPol Buffer, 1.8 µL of 8-HQ (500 µM) and 0.2 µL Therminator IX (NEB, 10 U/µL) were added, followed by a 10 min incubation at 60°C. 25 µL 8-HQ (500 µM) and 80 µL Pronex® beads were added for purification with an elution volume of 25 µL. To label oxidative modifications, 1 µL FPG (NEB, 8 U/µL), 1 µL ENDOIV (NEB, 10 U/µL) and 3 µL 10x NEBuffer 2 (NEB) were added for a final volume of 30 µL; the reaction mixture was incubated for 1 h at 37°C, immediately followed by adding 3.5 µL prop-dGTP (Jena Biosciences, 2.5 mM), 0.5 µL 10x NEBuffer 2 (NEB), 0.8 µL 8-HQ (500 µM) and 0.2 µL Therminator IX (NEB 10 U/µL) for 10-min incubation at 60°C. As for the plasmid DNA, each modified plasmid was separately spiked into 2 µg of the original pEGFP-W plasmid and subjected to these same initial steps. In the case of the uracil-modified plasmid, UDG (NEB, 5 U/µL) was used instead of FPG to excise the uracil base, and prop-dTTP (Jena Biosciences, 2.5 mM) was used instead of prop-dGTP. Two control samples containing 2 µg of unmodified pEGFP-W plasmid were processed under identical conditions with either FPG and prop-dGTP or UDG and prop-dTTP. Steps for mapping either AP sites or oxidative damage, both for gDNA and plasmid DNA, are identical from this point onwards. DNA was purified using Pronex® Magnetic Beads (Promega) with a sample:bead ratio of 50:80 and using an elution volume of 50 µL with Buffer EB. Sonication of DNA was performed using a Q800R2 sonicator (QSonica) with the following settings: 4°C, 20% amplitude, pulse 15 s on, 5 s off, total 5 min for gDNA and 4 min for plasmid DNA. The resulting DNA fragments were analyzed on a Tapestation 2200 using a High Sensitivity D100 ScreenTape (Agilent Technologies). DNA was purified using Pronex® beads with a sample:beads ratio of 1:2 and 50 µL elution volume. Samples were quantified using a Quantus™ Fluorometer. For adapter ligation, NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (NEB) was utilized using either 1 µg of gDNA or the remaining plasmid DNA and the pre-annealed NEB adapter (NEB_P7, NEB_P5, 15 µM; Supplementary Table 1). The manufacturer's instruction was followed without performing the USER enzyme step. The sample was purified using Pronex® beads in sample:beads ratio 1:2 and elution in 50 µL Buffer EB. Successful adapter ligation was checked on an Agilent TapeStation with a High Sensitivity D100 ScreenTape. The sample volume was then condensed to 6 µL using a vacuum concentrator. To denature DNA, the sample was heated to 95°C for 2 min and rapidly cooled on ice. CuAAC reaction was performed using 2 µL MoDIS (100 µM equimolar mixture of MoDIS_1 and MoDIS_2; Supplementary Table 1), 4 µL DMSO, 2 µL premixed CuSO4:THPTA (THPTA from Lumiprobe; 20 mM Cu2+ and 200 mM THPTA) and 2 µL sodium phosphate buffer (1 M, pH 7.0). The reaction was started by adding 2 µL sodium ascorbate (400 mM in H2O, freshly prepared) and incubated for 60 min at 37°C. Purification was done using Pronex® beads in a sample:beads ratio 1:2 and 30 µL elution volume. Biotin enrichment of sample DNA was performed using Dynabeads™ MyOne™ Streptavidin C1 (ThermoFisher). First, B&W buffer was prepared according to the manufacturer's protocol including 0.05 % Tween™ 20. Then, 15 µL Dynabeads™ were transferred to a fresh tube, washed three times with 200 µL 1x B&W buffer and resuspended in 30 µL 2x B&W buffer. The DNA sample was denatured by heating to 95°C for 2 min and rapid cooling on ice. Then, 30 µL sample was mixed with 30 µL of the prepared Dynabeads™ and rotated for 15 min at RT. Afterwards, the Dynabeads™ were washed 3x with 1x B&W buffer by resuspending the beads, 3 min rotation and 1 min pelleting on a magnetic rack. A final wash was performed with Buffer EB and Dynabeads™ were resuspended in 20 µL Buffer EB. To amplify the biotin-enriched material, a PCR was performed by adding 5 µL 10x ThermoPol buffer, 5 µL dNTP mix (NEB, 2 mM), 1 µL MgSO4 (100 mM), 1 µL ex_UMI (10 µM; Supplementary Table 1), 1 µL Pr_P7 (10 µM; Supplementary Table 1) and 0.5 µL Vent (exo-) DNA Polymerase (NEB 2 U/µL). The PCR program comprised an initial denaturation step (95°C, 120 s), followed by 5 cycles of denaturation (95°C, 20 s), annealing (59°C, 20 s) and elongation (72°C, 60 s) and a final elongation step (72°C, 300 s). The sample was purified with Pronex® beads in a sample:beads ratio 1:2 and an elution volume of 30 µL. Final library amplification and indexing was performed by adding 25 µL NEBNext® Ultra™ II Q5® Master Mix (NEB), 2.5 µL i5_Index (10 µM; Supplementary Table 1) and 2.5 µL i7_Index (10 µM; Supplementary Table 1) to 20 µL sample. The PCR program comprised an initial denaturation step (98°C, 30 s), 10 cycles of denaturation (98°C, 10 s), annealing (68°C, 30 s) and elongation (72°C, 20 s), and a final elongation step (72°C, 120 s). The same PCR setup can be used for a precedent qPCR by adding a qPCR dye as Evagreen (Biotium) to test for successful sample amplification. The PCR sample was purified with the Pronex® beads dual size selection protocol using an initial sample:beads ratio of 1:1, followed by a sample:beads ratio of 0.5:1 to exclude fragments longer than 1000 bp and shorter than 250 bp. The final sample concentration was adjusted to a concentration of 4 ng/µL and sequenced on an Illumina NovaSeq 6000.