Extended Data Fig. 1: Reporter silencing depends on DNMT3A and is concomitant with DNA methylation.

a, Schematic of lentiviral methylation reporter vectors. LTR, lentiviral long terminal repeat; TetO, tetracycline operator; SV40, simian virus 40 poly(A) sequence; rTetR, reverse Tet repressor. b, Representative gating scheme for flow cytometric analysis of reporter silencing assays. Helix NP NIR was used as a viability dye. Citrine fluorescence and mCherry fluorescence were monitored on the FITC and PE-Texas Red channels, respectively. c, Reporter methylation levels after varying duration of dox treatment measured by targeted bisulfite sequencing. In each plot, lines represent the percent cytosine methylation at each position (non-cytosine positions are set to 0). CpG sites are highlighted by dots. A schematic of the reporter is shown below, indicating the location of each sequenced region. This experiment was performed once (n = 1). d, Full timecourse for DNMT3-knockout silencing experiment shown in Fig. 1f (top), with representative histograms of citrine fluorescence from each day (bottom). e, Full timecourse for sgW698 silencing experiment shown in Fig. 1h (top), with representative histograms of citrine fluorescence from each day (bottom). f, Deep sequencing of cells edited with sgW698 after 9 days of dox treatment, either sorted for citrine⁺ cells (yellow) or unsorted (gray). Plot shows the percentage of aligned reads with C-to-T base edits at each indicated protospacer position, or the percentage of aligned reads with indels. Sequencing was performed once (n = 1). For d and e, data and error bars (where larger than data point) are mean ± s.d. of n = 3 replicates, and results are representative of two independent experiments.

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