Data related to "Structural and biochemical insight into the mechanism of dual CpG site binding and methylation by DNMT3A"

Methylation of substrate libraries Single-stranded DNA oligonucleotides used for generation of double stranded substrates with different distance between CpG sites were obtained from IDT. Sixteen single-stranded oligonucleotides were pooled in equimolar amounts and the second strand synthesis was conducted by a primer extension reaction using one universal primer. The obtained mix of double-stranded DNA oligonucleotides was methylated by DNMT3A catalytic domain and DNMT3A/3L and incubated for 60 min at 37 °C in the presence of 0.8 mM S-adenosyl-L-methionine (Sigma) in reaction buffer (20 mM HEPES pH 7.5, 1 mM EDTA, 50 mM KCl, 0.05 mg/mL bovine serum albumin). For DNMT3A, concentrations of 0.25 µM, 0,5 µM, 1 µM and 2 µM were used, for DNMT3A/3L 0.125 µM and 0.25 µM. In addition, a no-enzyme control was processed identically to all other samples. Reactions were stopped by shock freezing in liquid nitrogen, then treated with proteinase K for 2 hours at 42 °C. Afterwards DNA was digested with BsaI-HFv2 enzyme and a hairpin (pGAGAAGGGATGTGGATACACATCCCT) was ligated using T4 DNA ligase (NEB). DNA was bisulfite converted using EZ DNA Methylation-Lightning kit (ZYMO RESEARCH) according to the manufacturer protocol, purified and eluted with 10 µL ddH2O. NGS library generation Libraries for Illumina Next Generation Sequencing (NGS) were produced with the two-step PCR approach. In the first PCR, 2 µL of bisulfite-converted DNA were amplified with the HotStartTaq DNA Polymerase (QIAGEN) and primers containing internal barcodes using following conditions: 15 min at 95 °C, 10 cycles of 30 sec at 94 °C, 30 sec at 50 °C, 1 min and 30 sec at 72 °C, and final 5 min at 72 °C; using a mixture containing 1x PCR Buffer, 1x Q-Solution, 0.2 mM dNTPs, 0.05 U/µL HotStartTaq DNA Polymerase, 0.4 µM forward and 0.4 µM reverse primers in a total volume of 20 µL. In the second PCR, 1 µL of obtained products were amplified by Phusion Polymerase (Thermo) with another set of primers to introduce adapters and indices needed for NGS (30 sec at 98 °C, 10 cycles - 10 sec at 98 °C, 40 sec at 72 °C, and 5 min at 72 °C). PCRII was carried out in 1x Phusion HF Buffer, 0.2 mM dNTPs, 0.02 U/µL Phusion HF DNA Polymerase, 0.4 µM forward and 0.4 µM reverse primers in a total volume of 20 µL. Obtained libraries were pooled in equimolar amounts, purified and sequenced in the Max Planck Genome Centre Cologne. Bioinformatic analysis Bioinformatic analysis of obtained NGS data was conducted with a local Galaxy server and with home written scripts. Briefly, fastq files were analyzed by FastQC, 3’ ends of the reads with a quality lower than 20 were trimmed and reads containing both full-length sense and antisense strands were selected. Next, the samples were split using the internal barcodes with respect to the different experimental conditions. Afterwards the insert DNA sequence was extracted and used for further downstream analysis. The uploaded text files contain the bisulfite converted sequences with pairs of CpG sites in variable distance as described in the furhter documentation (info.pdf).

底物库的甲基化：用于生成具有不同CpG位点间距离的双链底物的单链DNA寡核苷酸，由IDT公司提供。将十六种单链寡核苷酸以等摩尔比例混合，并使用一种通用引物通过引物延伸反应合成第二链。所得的双链DNA寡核苷酸混合物经DNMT3A催化域和DNMT3A/3L催化甲基化，并在37°C下含有0.8 mM S-腺苷-L-蛋氨酸（Sigma）的反应缓冲液（20 mM HEPES pH 7.5，1 mM EDTA，50 mM KCl，0.05 mg/mL牛血清白蛋白）中孵育60分钟。对于DNMT3A，使用0.25 µM、0.5 µM、1 µM和2 µM的浓度，对于DNMT3A/3L使用0.125 µM和0.25 µM。此外，无酶对照组的处理方式与其他所有样本相同。通过在液氮中急速冷冻终止反应，然后于42°C下用蛋白酶K处理2小时。之后，使用BsaI-HFv2酶消化DNA，并使用T4 DNA连接酶（NEB）将发夹结构（pGAGAAGGGATGTGGATACACATCCCT）连接。DNA使用EZ DNA Methylation-Lightning试剂盒（ZYMO RESEARCH）按照制造商协议进行亚硫酸氢盐转化，纯化后用10 µL ddH2O洗脱。Illumina下一代测序（NGS）文库的生成：采用两步PCR方法制备Illumina NGS文库。在第一步PCR中，使用HotStartTaq DNA聚合酶（QIAGEN）和包含内部条码的引物将2 µL亚硫酸氢盐转化的DNA扩增，条件如下：95°C 15分钟，94°C 30秒，50°C 30秒，72°C 1分30秒，循环10次，最后72°C 5分钟；使用含有1x PCR Buffer，1x Q-Solution，0.2 mM dNTPs，0.05 U/µL HotStartTaq DNA聚合酶，0.4 µM正向和0.4 µM反向引物，总体积20 µL的混合物。在第二步PCR中，使用Phusion聚合酶（Thermo）和另一套引物将1 µL所得产物扩增，以引入NGS所需的适配器和索引（98°C 30秒，98°C 10次循环，40秒于72°C，5分钟于72°C）。PCRII在1x Phusion HF Buffer，0.2 mM dNTPs，0.02 U/µL Phusion HF DNA聚合酶，0.4 µM正向和0.4 µM反向引物，总体积20 µL中进行。所得文库以等摩尔比例混合，纯化并在科隆马克斯普朗克基因组中心进行测序。生物信息学分析：使用本地Galaxy服务器和自编脚本对获得的NGS数据进行生物信息学分析。简要来说，通过FastQC分析fastq文件，去除质量低于20的3'端读取，选择包含全长感性和反义链的读取。然后，根据不同的实验条件使用内部条码将样本分开。之后，提取插入DNA序列，用于后续分析。上传的文本文件包含亚硫酸氢盐转化的序列，其中CpG位点对之间的距离如进一步文档（info.pdf）所述。