Abstract
Method overview: For targeted DNA demethylation of the H19/IGF2 ICR1, HEK293 cells were transfected with two plasmids, one containing dCas9 fused to a SunTag with five repeats of the GCN4 peptide, separated by 22 aa long linkers, and scFv-fused TET1CD, as well as a GFP reporter protein. The second plasmid encodes five sgRNAs targeting the ICR1 and a DsRed fluorophore. On day 3 post-transfection, GFP- and DsRed-positive cells were sorted by FACS. A part of the sorted cells was used immediately for downstream analysis, the other part was re-seeded to harvest at later time points. Genomic DNA was isolated from the cells and bisulfite or oxidative bisulfite conversion was conducted. For amplicon-based DNA methylation analysis, libraries were prepared from bisulfite-converted DNA using two consecutive PCRs in which barcodes, indices and sequencing adapters are added. Samples were sequenced by NGS and data was analyzed. Method details: The gDNA of transfected HEK293 cells sorted by FACS was isolated using the QIAmp DNA Mini Kit (Qiagen) according to the manufacturer's instructions. 500 ng gDNA was fragmented enzymatically by overnight digestion using 40 U EcoRV-HF (a non-cutter in the genomic regions desired for amplification) (New England BioLabs, Inc.) in CutSmart buffer in a total volume of 20 µl. The next day, bisulfite conversion was conducted using the EZ DNA Methylation-Lightning™ Kit (ZYMO RESEARCH) according to the manufacturer's protocol. Oxidative bisulfite conversion was performed using the TrueMethyl® oxBS Module (Part No. 0414, Tecan Genomics, Inc.) according to the manufacturer's instructions. Amplicons of interest were amplified in a first PCR1 with locus-specific primers, which also contained barcodes and adapters complementary to PCR2 primers. The PCR1 product was used as template for PCR2, in which Illumina TruSeq sequencing indices are added to the amplicons. Sample concentrations were measured using the NanoDrop 1000 (Thermo Fisher Scientific) and equimolar amounts of samples were pooled. Paired-end Illumina sequencing with 250 bp read length was performed by Novogene (UK) Company Limited. Data analysis: NGS data in a FASTQ format was analyzed basically as described (Rajaram et al., 2023) on the Galaxy platform (https://usegalaxy.org/) (The Galaxy platform for accessible, reproducible and collaborative biomedical analyses, 2022), where all the following tools are available. In brief, Illumina adapter sequences were removed using Trim Galore!. Afterwards, two paired-end reads were merged using Pear and reads with low quality were removed with Filter FASTQ. De-multiplexing of individual samples tagged with combinations of barcodes and Illumina indices was done by converting the FASTQ files using FASTQ to Tabular, followed by selection of lines with the tool Select and re-conversion of the files to a FASTQ format with Tabular to FASTQ. For the alignment of reads to a reference sequence, bwameth was used and the DNA methylation at each CpG site was analyzed by applying the tool MethylDackel. The output files were processed using Microsoft Excel. References: The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2022 update. Nucleic acids research 2022, 50, W345-W351, doi: 10.1093/nar/gkac247Rajaram, N.; Kouroukli, A.G.; Bens, S.; Bashtrykov, P.; Jeltsch, A. Development of super-specific epigenome editing by targeted allele-specific DNA methylation. Epigenetics Chromatin 2023, 16, 41, doi: 10.1186/s13072-023-00515-5
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