N2-heptynyl-dG sequencing

N2-heptynyl-dG sequencing in HEK293T cells HEK293T cells were treated with N2-heptynyl-dG for 3-h at a final concentration of 10 μM. The cells were then harvested immediately for N2-heptynyl-dG sequencing. The genomic DNA were extracted using Monarch® Genomic DNA Purification Kit (New England Biolabs) and reacted with biotin-azide at room temperature for 6 h. The biotinylated genomic DNA was sheared using a Covaris S220 sonicator. The N2-heptynyl-dG DNA were enriched with Dynabeads™ MyOne™ Streptavidin C1 (Thermo). Purified DNA was quantified and verified on an Agilent 2100 Bioanalyzer. The library was then constructed using NEBNext Ultra DNA Library Prep Kit (NEB, E7103S) following the manufacturer’s instructions. Subsequently, the purified DNA libraries were assessed using an Agilent 2100 Bioanalyzer and multiplexed for sequencing on a DNBSEQ-G400 (BGI Genomics) .HEK293T细胞中N2-庚炔基-dG（N2-heptynyl-dG）的测序实验：将HEK293T细胞以终浓度10 μM的N2-庚炔基-dG处理3小时，随后立即收获细胞用于该测序实验。使用Monarch®基因组DNA纯化试剂盒（Monarch® Genomic DNA Purification Kit，New England Biolabs，新英格兰生物实验室）提取基因组DNA，于室温下与生物素叠氮（biotin-azide）反应6小时。采用Covaris S220超声破碎仪对生物素标记的基因组DNA进行片段化。使用Dynabeads™ MyOne™ 链霉亲和素C1磁珠（Dynabeads™ MyOne™ Streptavidin C1，Thermo，赛默飞世尔科技）富集携带N2-庚炔基-dG的DNA片段。通过Agilent 2100生物分析仪（Agilent 2100 Bioanalyzer）对纯化后的DNA进行定量与质量验证。按照制造商的操作指南，使用NEBNext Ultra DNA文库制备试剂盒（NEBNext Ultra DNA Library Prep Kit，NEB, E7103S）构建测序文库。再次通过Agilent 2100生物分析仪对纯化后的测序文库进行质量评估，随后进行多重索引标记，并在DNBSEQ-G400测序平台（DNBSEQ-G400，BGI Genomics，华大基因）上完成测序。