GSM7815875: SP1_MUT_Bio1; 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 µL TAPS buffer and resuspended in 0.5 mg/mL Proteinase K, 0.5% SDS in 10 mM Tris-HCl buffer pH 8.0 at 55°C for 1h at 800 rpm. Tagmented DNA was purified with DNA clean & concentrator columns (Zymo Research, D4013). For Illumina library preparation, genomic DNA extracted by CUT&Tag was used. Libraries were generated as previously described with a universal i5 and a uniquely barcoded i7 primer(Kaya-Okur, H.S., Wu, S.J., Codomo, C.A. et al. Nat Commun 10, 1930 (2019)). Libraries were visualized and quantified on a high-sensitivity D1000 tape on a Tape Station (AgilentTechnologies 2200)andwith the NEBNext®Library Quant Kit for Illumina (NEB, cat#7630S), following the manufacturer's instructions.Libraries were sequenced on an Illumina NextSeq 500 (High Output Kit v2.5, 75 cycles) or NextSeq 2000 instrument(P2 or P3 reagents, 100 cycles). RNA-seq and library preparation Total RNA was extracted from 1.0x106 cells using the RNeasy Plus Mini Kit (Qiagen, cat#74134) as per manufacturer's instructions. RNA quality was checked via TapeStation (Agilent Technologies 2200, RIN>9) and RNA concentration determined using a Qubit fluorometer (Thermo Fisher, Qubit 4). Libraries were generated from 700ng RNA using the NEBNext®Poly(A) mRNA Magnetic Isolation Module (NEB, cat#7490S) together with the NEBNext®UltraTMII Directional RNA Library Prep Kit for Illumina (NEB, cat#7760S) as per manufacturer's instructions. Libraries were sequenced on a NextSeq 500 system (High Output Kit v2.5, 75 cycles). For MNase 1.0 x 106 cells were washed in ice-cold PBS and incubated in 1mL lysis buffer (10 mM Tris-HCl pH 8.0, 60 mM KCl, 15 mM NaCl, 3 mM MgCl2, 1 mM DTT, 0.1% NP-40) for 10 mins on ice. Cells were then centrifuged at 1,000 x g for 5 mins at 4 °C and resuspended in lysis buffer without NP-40 (10 mM Tris-HCl pH 8.0, 60 mM KCl, 15 mM NaCl, 3 mM MgCl2, 1 mM DTT). Cells were then incubated at 37 °C for 5 mins supplemented with 2 mM CaCl2 and digested with 10 U MNase at 37 °C for 60 mins. The digestion was stopped with a final concentration of 40 mM EDTA, 0.5% SDS. DNA was incubated with RNase A (Thermo Scientific, cat #EN0531) for 1 h at 37oC. DNA was purified using Phenol-chloroform-isoamyl alcohol (PCl, Thermo Scientific, cat #15593049). Libraries were generated with the NEBNext® UltraTM II Directional RNA Library Prep Kit for Illumina (NEB, cat #7760S) as per manufacturer's instructions. A bead-based size selection of 200 bp was performed to isolate mononucleosomal fragments. Libraries were then quantified with the NEBNext® Library Quant Kit for Illumina (NEB, cat #7630S) and sequenced on a NextSeq 500 system (High Output Kit v2.5, 75 cycles).