design: Library strategy: CUT&TAG. R loop profiles were captured by the S9.6 CUT&Tag method as established previously (Wang et al., 2021) and using a CUT&Tag kit (Active Motif, 53165) according to the manufacturer's instructions. Briefly, 1x106 cells were harvested and washed twice in ice cold Wash Buffer (20mM HEPES pH 7.5, 150mM NaCl, 0.5mM spermidine) supplemented with protease inhibitors. Concanavalin A beads were added into the cell suspension and rotated at room temperature for 10 minutes. Bead-bound cells were then separated from the supernatant on a magnetic rack, and cells were resuspended in ice cold Antibody Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.05% digitonin, 0.01% NP-40, and 2 mM EDTA) supplemented with protease inhibitors. S9.6 antibody (2g) (Kerafast, ENH001) was added into bead-bound cells and rotated at 4oC overnight, with negative control samples supplemented with 10U RNaseH (NEB, M0297L). After overnight incubation, the secondary antibody diluted in Dig-Wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.05% digitonin, and 0.01% NP-40) supplemented with protease inhibitors was added and rotated at room temperature for 1 hour. The bead-bound cells were then washed in Dig-Wash buffer three times. pA-Tn5 Transposomes were then diluted in Dig-300 buffer (20 mM Hepes (pH 7.5), 300 mM NaCl, 0.5 mM spermidine, and 0.05% digitonin) supplemented with protease inhibitors, and incubated with rotation with the bead-bound cells for 1 hour at room temperature. Excess transposomes were removed by three washed with Dig-300 buffer. Finally, samples were tagmented in 125l Tagmentation Buffer (10 mM TAPS-NaOH pH 8.5, 10 mM MgCl2, 10% DMF, and 0.85 mM ATP) at 37oC for 1 hour. To stop the tagmentation reaction, 4.2l 0.5M EDTA, 1.25l 10% SDS and 1.1l 10mg/ml ProteinaseK was added and samples were incubated at 55oC for 1 hour to solubilize DNA fragments before column purification. DNA samples were then eluted and libraries were amplified using a combination of Illumina i7/i5 indexed primers. Library cleanup was performed with a preliminary 1.1x SPRI bead cleanup followed by size selection with 0.5x and 1x Ampure XP beads (Beckman Coulter, A63880). Libraries were then subjected to fragment analysis and Illumina sequencing.