NGS data related to Rajaram et al.: Development of super-specific epigenome editing by targeted allele-specific DNA methylation

Method overview To achieve targeted ASM, transient transfection of the dCas9-10X SunTag-BFP, scFv-DNMT3A-3L-sfGFP, and sgRNA-DsRed plasmids was performed in HEK293 cells. Control experiments were conducted with a scrambled sgRNA that does not have a binding site in the human genome. Initial studies showed that cells positive for all three plasmids exhibited highest fluorescence of the corresponding reporter proteins on day 3 post-transfection. Hence FACS sorting was conducted at this time point. Genomic DNA was isolated from the FACS-sorted cells at day 3 after transfection and subjected to bisulfite treatment. Library preparation was performed using the bisulfite-converted samples, followed by NGS and data analysis. Most methylation experiments were conducted in three independent biological replicates. Method details Genomic DNA was extracted using QIAmp DNA Mini Kit (Qiagen). 500 ng of genomic DNA was subjected to overnight digestion with EcoRV which is not cutting in any of the target amplicons. Zymo EZ DNA Methylation-Lightning Kit (D5030-E) was used for bisulfite conversion. The library for NGS was prepared by two consecutive PCR reactions (Leitao et al, 2018). Firstly, bisulfite converted genomic DNA of each sample was amplified with target gene specific primers. The gene specific optimized amount of a product from the first PCR was used as a template for the second PCR to add the Illumina TruSeq sequencing adapters. Final products were quantified, pooled in equimolar amounts and purified using SPRIselect beads (Beckman Coulter). Ready-to-use pools of libraries were sequenced on NovaSeq 6000 using a PE250 flow cell (Novogene). NGS data were obtained in the form of FASTQ files. Data analysis FASTQ files were processed on the local instance of Galaxy server as described earlier (Bashtrykov & Jeltsch, 2018) with some modifications. Briefly, an adaptor and low-quality trimming was conducted using Trim Galore! (developed by Felix Krueger at the Babraham Institute). The two associated paired reads were merged using PEAR (Zhang et al, 2014). Experiment specific combinations of Illumina indices and barcodes were used to extract reads for the individual experiments from the pool of reads. This information is provided in the included Excel file. Reads corresponding to different alleles of the same target gene were identified based on the presence of the SNP. Two files of reads corresponding to alleles were generated and their methylation level was analyzed independently. First, reads were mapped against a reference sequence of the target region using bwameth (Pedersen et al, 2014) and then methylation of individual CpG sites was computed using MethylDackel (https://github.com/dpryan79/MethylDackel). Final visualization and statistics were prepared using Microsoft Excel. References Bashtrykov P, Jeltsch A (2018). DNA Methylation Analysis by Bisulfite Conversion Coupled to Double Multiplexed Amplicon-Based Next-Generation Sequencing (NGS). Methods Mol Biol 1767: 367-382 Leitao E, Beygo J, Zeschnigk M, Klein-Hitpass L, Bargull M, Rahmann S, Horsthemke B (2018). Locus-Specific DNA Methylation Analysis by Targeted Deep Bisulfite Sequencing. Methods Mol Biol 1767: 351-366 Pedersen JS, Valen E, Velazquez AM, Parker BJ, Rasmussen M, Lindgreen S, et al., Orlando L (2014). Genome-wide nucleosome map and cytosine methylation levels of an ancient human genome. Genome Res 24: 454-466 Zhang J, Kobert K, Flouri T, Stamatakis A (2014). PEAR: a fast and accurate Illumina Paired-End reAd mergeR. Bioinformatics 30: 614-620

方法概述：为达成特定表型筛选（ASM），本研究在HEK293细胞中进行了dCas9-10X SunTag-BFP、scFv-DNMT3A-3L-sfGFP及sgRNA-DsRed质粒的瞬时转染。对照组实验使用了不具有结合位点的随机sgRNA。初步研究表明，在转染后第3天，所有三种质粒阳性的细胞展现出相应报告蛋白的最高荧光强度。因此，在此时点进行了流式细胞术分离。在转染后第3天，从流式分离的细胞中提取基因组DNA，并进行了亚硫酸氢盐处理。利用亚硫酸氢盐转化样本制备文库，随后进行高通量测序（NGS）及数据分析。大多数甲基化实验均在三个独立的生物学重复中完成。
方法细节：采用QIAmp DNA Mini Kit（Qiagen）提取基因组DNA。500 ng的基因组DNA在过夜消化过程中使用EcoRV酶，该酶在所有目标扩增子中均不切割。使用Zymo EZ DNA Methylation-Lightning Kit（D5030-E）进行亚硫酸氢盐转化。NGS文库的制备通过连续两次PCR反应完成（Leitao等，2018年）。首先，每个样本的亚硫酸氢盐转化基因组DNA使用针对目标基因的特异性引物进行扩增。第一次PCR产生的基因特异性优化产物的量作为第二次PCR的模板，以添加Illumina TruSeq测序适配器。最终产物经过定量，等摩尔混合，并使用SPRIselect磁珠（Beckman Coulter）纯化。准备好的文库池在NovaSeq 6000测序仪上使用PE250流式细胞器进行测序（Novogene）。NGS数据以FASTQ文件的形式获得。
数据分析：在本地Galaxy服务器实例上对FASTQ文件进行处理，如前所述（Bashtrykov & Jeltsch，2018年），并进行了一些修改。简而言之，使用Trim Galore!（由Felix Krueger在Babraham研究所开发）进行适配器和低质量序列的修剪。使用PEAR（Zhang等，2014年）合并两个关联的配对读段。根据SNP的存在识别对应同一目标基因不同等位基因的读段。生成两个对应等位基因的读段文件，并独立分析其甲基化水平。首先，使用bwameth（Pedersen等，2014年）将读段映射到目标区域的参考序列上，然后使用MethylDackel（https://github.com/dpryan79/MethylDackel）计算单个CpG位点的甲基化。最终的可视化和统计分析使用Microsoft Excel完成。
参考文献：Bashtrykov P, Jeltsch A (2018). DNA Methylation Analysis by Bisulfite Conversion Coupled to Double Multiplexed Amplicon-Based Next-Generation Sequencing (NGS). Methods Mol Biol 1767: 367-382. Leitao E, Beygo J, Zeschnigk M, Klein-Hitpass L, Bargull M, Rahmann S, Horsthemke B (2018). Locus-Specific DNA Methylation Analysis by Targeted Deep Bisulfite Sequencing. Methods Mol Biol 1767: 351-366. Pedersen JS, Valen E, Velazquez AM, Parker BJ, Rasmussen M, Lindgreen S, et al., Orlando L (2014). Genome-wide nucleosome map and cytosine methylation levels of an ancient human genome. Genome Res 24: 454-466. Zhang J, Kobert K, Flouri T, Stamatakis A (2014). PEAR: a fast and accurate Illumina Paired-End reAd mergeR. Bioinformatics 30: 614-620.