Targeted bisulfite sequencing of TagRFP reporter (DNMT3B_+doxON)
HEK293T reporter cells stably expressing rTetR-antiDNMT1, rTetR-DNMT1, or rTetR-DNMT3B were treated with 1 g/mL doxycycline for 5 days. Afterwards, each treated cell line was sorted in TagRFP negative (+dox OFF) and TagRFP positive (+dox ON) cells using a SONY SH800 Cell Sorter. Total genomic DNA was extracted from these cells. As a control, we also extracted DNA from cells not treated with dox (-dox) grown at the same time as the +dox cells for each cell line and sorted to include the entire population. Assays were designed targeting CpG sites in 100-300 nucleotide regions at the TagRFP reporter gene and two control genes, IGF2 (positive) and ACTB (negative). Amplification of all samples using region-specific primer pairs was performed; the resulting amplicons were pooled and subsequently barcoded using PCR. After barcoding, samples were purified and then prepared for massively parallel sequencing using a MiSeq V2 300bp Reagent Kit (Illumina) and paired-end sequencing protocol according to the manufacturers guidelines. Sequence reads were identified using standard Illumina base-calling software.