GSM7815907: TPL_RNAPIIS5P_WT_30m_B3; Homo sapiens; OTHER
CUT&Tag and Chem-map were performed as previously described (Kaya-Okur, H.S., Wu, S.J., Codomo, C.A. et al. Nat Commun 10, 1930 (2019),Yu, Z., et al. Nat Biotechnol (2023)).G4 mapping (BG4, SG4) and R-loop profiling were performed as 3-layer CUT&Tag on DynabeadsTMMyOneTMStreptavidinT1 (Thermo Fisher, cat#65601). PDS Chem-map, G4 interactome CUT&Tag experiments (SP1, CNBP, MLL1, MLL4, SIN3A, SMARCA4, RNAPIIS5P, RNAPIIS2P) and histone marks (H3K4me1, H3K4me3, H3K27me) were performed as 2-layer CUT&Tag on ConcanavalinA (ConA)-coated magnetic beads (Bangs Laboratories, BP531). MyOneT1 beads were washed twice with PBS pH 6.8, resuspended in 1xPBS pH 6.8 supplemented with 0.01% Tween-20 and mixed with the biotin-conjugated ConA (Sigma-Aldrich, cat#C5275-5MG) solution at 2.3mg/mL at a 1:0.5 ratio. The mix was then incubated at 22°C at 400 rpm for 30min. Aliquots of 500,000 cells/100mL were added to 10mL of beads, washed twice with 100mL wash buffer and resuspended in 1% BSA Antibody Buffer. Primary antibodies were added (1:50), and the mix was incubated at 4°C overnight at 600 rpm. Cells were then washed twice with 100mL dig-wash buffer (0.05% digitonin, 20 mM HEPES pH 7.5, 150 mM KCl, 0.5 mM spermidine in nuclease-free water with a Roche Complete Protease Inhibitor EDTA-free tablet) and resuspended in 50mL dig-wash buffer. Secondary antibodies were added 1:50 to the samples and incubated at 25°C for 2h at 600 rpm (2-layer CUT&Tag). For G4-mapping samples, cells were washed three times with 500mL dig-wash buffer and resuspended in 50mL dig-wash buffer and 0.5mL of guinea-pig anti-rabbit antibody (Antibodies online, ABIN101961)was added and incubated with the cells at 25°C for 1h at 600 rpm (3-layer CUT&Tag). Cells were then washed three times with 100mL Dig-300 buffer (0.01% digitonin, 20mM HEPES pH7.5, 300mM KCl, 0.5mM spermidine in nuclease-free water with a Roche Complete Protease Inhibitor EDTA-free tablet) and incubated in 1:250 dilution of pA-Tn5 in 50µL Dig-300 buffer at 25°C for 1h at 600rpm.Cells were then washed three times with 500 µL Dig-300 buffer and the Tn5 tagmentation was activated by incubating in 300mL Dig-300 buffer supplemented with 10 mM MgCl2at 37°C for 1h at 600 rpm. Cells were then washed twice with 500 µ