Data related to "Preferential interaction of DNMT3A subunits containing the R882H cancer mutation leads to dominant changes of flanking sequence effects"

Methylation of substrate libraries Single-stranded DNA oligonucleotides used for generation of double stranded substrates with a distance of 12 base pairs between CpG sites were obtained from IDT. Second strand synthesis was conducted by a primer extension reaction using one universal primer. The obtained library of double-stranded DNA oligonucleotides was methylated by different purified heterotetramers containing DNMT3A catalytic domain and DNMT3A R882H catalytic domain subunit, boht either containing a His-tag or MBD-tag. For this it was 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). DNA concentrations were 107 nM, DNMT3A concentrations were used between 0.05 and 0.1 µM. 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 12 bp distance as described in the furhter documentation (info.pdf). The naming of the files is as follows: Name, Complex, Repeat, c(DNMT3AC) [µM] 1WW, His-WT/MBP-WT, R1, 0.1 2WW, His-WT/MBP-WT, R2, 0.05 1RR, His-R882H/MBP-R882H, R1, 0.1 2RR, His-R882H/MBP-R882H, R2, 0.07 1RW, His-R882H/MBP-WT, R1, 0.1 2RW, His-R882H/MBP-WT, R2, 0.1 1WR, His-WT/MBP-R882H, R1, 0.1 2WR, His-WT/MBP-R882H, R2, 0.1

底物文库的甲基化：用于生成间隔12个碱基对的二链底物的单链DNA寡核苷酸从IDT公司获取。第二链合成通过使用通用引物进行引物延伸反应完成。获得的二链DNA寡核苷酸文库经不同纯化的异四聚体甲基化，这些异四聚体包含DNMT3A催化域和DNMT3A R882H催化域亚基，其中既包含His标签也包含MBD标签。为此，将其在37°C下与0.8 mM S-腺苷-L-蛋氨酸（Sigma）在反应缓冲液（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。DNA浓度为107 nM，DNMT3A浓度为0.05至0.1 µM。通过在液氮中迅速冷冻终止反应，然后在42°C下用蛋白酶K处理2小时。之后，用BsaI-HFv2酶消化DNA，并使用T4 DNA连接酶（NEB）将发夹（pGAGAAGGGATGTGGATACACATCCCT）连接起来。使用EZ DNA Methylation-Lightning试剂盒（ZYMO RESEARCH）根据制造商协议进行亚硫酸氢盐转化，纯化，并以10 µL ddH2O洗脱。Illumina下一代测序（NGS）文库生成：采用两步PCR方法生产Illumina下一代测序（NGS）文库。在第一步PCR中，使用HotStartTaq DNA聚合酶（QIAGEN）和包含内部条形码的引物将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，10个循环，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反向引物的混合物，总体积为20 µL。在第二步PCR中，使用Phusion聚合酶（Thermo）和另一套引物将1 µL获得的产品扩增，以引入NGS所需的适配器和索引（98°C 30秒，10个循环 - 98°C 10秒，72°C 40秒，72°C 5分钟）。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反向引物的总体积为20 µL的条件下进行。获得的文库按等摩尔量混合，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。简而言之，通过FastQC分析fastq文件，剪除质量低于20的3'端读段，并选择包含全长正链和反链的读段。随后，根据不同的实验条件使用内部条形码将样本分割。之后，提取插入DNA序列并用于后续分析。上传的文本文件包含12 bp距离处的成对CpG位点的亚硫酸氢盐转化序列，如进一步文档（info.pdf）所述。文件命名如下：名称，复杂度，重复序列，c(DNMT3AC) [µM] 1WW，His-WT/MBP-WT，R1，0.1 2WW，His-WT/MBP-WT，R2，0.05 1RR，His-R882H/MBP-R882H，R1，0.1 2RR，His-R882H/MBP-R882H，R2，0.07 1RW，His-R882H/MBP-WT，R1，0.1 2RW，His-R882H/MBP-WT，R2，0.1 1WR，His-WT/MBP-R882H，R1，0.1 2WR，His-WT/MBP-R882H，R2，0.1