GSM8152784: SLAM-seq_U2OS_G4switch_dark_rep3; Homo sapiens; OTHER
Cells were washed twice in 200 μl TAPS wash buffer (10 mM TAPS, 0.2 mM EDTA), and resuspended in 100 μl extraction buffer (10 mM Tris HCl, pH 8.0, 0.5 mg/ml proteinase K (Thermo Scientific, catalogue no. EO0491), 0.5% SDS) with vortexing, and incubated at 55 ℃ for 1.5 h at 800 rpm. DNA extraction was performed using the DNA Clean & Concentrator-5 kit (Zymo Research, catalogue no. D4013). DNA was eluted in 25 μl provided DNA Elution Buffer from the column after incubation at room temperature for 10 min. To amplify DNA libraries, PCR was performed by mixing 21 μl tagmented DNA with 2 μl uniquely barcoded v2 Ad1.x primer (10 μM) and 2 μl uniquely barcoded v2 Ad2.x primer (10 μM)51, and 25 μl NEBNext Ultra II Q5 2× PCR Master mix (New England Biolabs, catalogue no. M0544) using the following cycle: 72 ℃ for 5 min; 98 ℃ for 30 s; 10 cycles of 98 ℃ for 10 s and 63 ℃ for 10 s; 72 ℃ for 1 min. Libraries were cleaned up by adding 1.3× volume (65 μl) of Ampure XP beads (Beckman Coulter, catalogue no. A63882), incubated at room temperature for 10 min, washed twice gently in 200 μl of 80% ethanol. DNA was eluted in 25 μl of 10 mM Tris HCl, pH 8.0. The size distribution of libraries was confirmed by TapeStation analysis using High Sensitivity D1000 ScreenTape (Agilent, catalogue no. 5067–5584). The libraries were quantified using the NEBNext Library Quant Kit (New England Biolabs, catalogue no. E7630L). Libraries were balanced and pooled, followed by size selection by adding 0.4× volumn of Ampure XP beads and incubation at room temperature for 10 min. The supernatant was transferred to a new DNA LoBind tube, and 1.3× volumn of Ampure XP beads was then added and incubated at room temperature for 10 min. On-bead libraries were washed twice with 80% ethanol and eluted in 30 μl of 10 mM Tris HCl, pH 8.0. Paired-end sequencing of libraries was performed on an NextSeq 2000 sequencer (Illumina) using the NextSeq 1000/2000 P2 reagent kit (100 cycles) (Illumina, catalogue no. 20046811). For chem-map competition experiments, U2OS cells were grown to a 70~80% confluence until treatment with PDS (4 μM or 20 μM) or vehicle DMSO at 37 ℃ for 3 h. Cells were washed twice with PBS, followed by performing standard G4switch Chem-map protocol.