GSM5665719: ES B6xJF1 Rep2 2; Mus musculus; OTHER
For G4 access: Trypsinized Haacht cells, resuspended S2 cells and suspension of K562, Raji and mouse ESC cells were pelleted and rinsed twice in phosphate-buffered saline buffer (PBS). Haacht cells were suspended in 5 mL of permeabilization buffer (150 mM of sucrose, 80 mM Kill, 5 mM KH2PO4, 5 mM MgCl2, 0.5 mM CaCl2 and 35 mM HEPES pH 7.4) and homogenized using a Dounce (40 strokes). For MNase digestion, 5x10^6 cells per titration point were resuspended in 50 μL of prewarmed permeabilization buffer supplemented with 0.2% (v/v) NP40 and incubated 5 minutes at 37ºC. Digestion took place by adding a volume of 500 mL of prewarmed MNase reaction buffer (150 mM sucrose, 50 mM Tris-HCl pH 8, 50 mM NaCl and 2 mM CaCl2) to each reaction. Either 3.1, 6.2, 12.5, 25 or 50U MNase (Merck, 10107921001) were added to the MNase reaction buffer and incubated at 37 ºC for 10 min. Digestion was stopped on ice and by adding 11 μL of 500 mM EDTA to each reaction. The digestions were incubated 10 minutes on ice with 550 μL of SDS lysis buffer (1% (v/v) SDS, 10 mM EDTA and 50 mM Trish pH 8) and 1 mL of water. Before DNA purification, digestions were incubated with 5 mL of RNaseq A (Thermofisher, EN0531) at 37 ºC for 2 hours and with 8 mL of proteinase K (Euromoney, 09-0911) at 56 ºC for 2 hours. To verify the performance of the digestion, 125 μL of each sample was cleaned-up using QI quick PCR Purification Kit (QIAGEN, 28106). Digestions were assessed by agarose gel and Bioanalyzer (Fig. 1c). The remaining digestions were purified by phenol-chloroform and ethanol precipitation for subsequent steps. Yeast S288C were pelleted and rinsed twice in phosphate-buffered saline buffer (PBS). Pellets from 100mL culture (OD: 0.5) were suspended in 600ul cell breaking buffer (20% glycerol, 100mM Tris pH 7.5), 600µl zirconia beads (0.5mm), 10µl of 100x protease inhibitors (Roche, 06538282001). Beads beating was performed in 1.5mL screw cap tubes in a Bullet Blender (Next advance) for 4 x 3min at a strength of 8 in a cold room. Cell suspensions were recovered by puncturing the bottom of tube (23 gauge syringes) and spined at 170 ruff for 3min into a 5mL Eppendorf tube. Everything was transferred into 1.5mL Eppendorf, then centrifuged for 5min at max speed and the top layers were discarded. Samples were suspended in 300ul of prewarmed NP buffer (50mM NaCl, 10mM Tris pH 7.4, 5mM MgCl2, 1mM CaCl2, 0.2% NP40 (v/v), 0.5mM spermidine (Sigma, S0266-1G), 0.007% B-Mercaptothion (Sigma, M3148-100ML). Digestion took place by adding a volume of 300µL prewarmed NP buffer supplemented with either 60u, 30u, 15u, 7.5u, 3.75, 1.9u, 1u of MNase (Merck 10107921001). Digestions were stopped on ice and by adding 150μL of stop buffer (5% SDS, 50mM EDTA). Before DNA purification, digestions were incubated with 5 µL of RNaseq A (Thermofisher, EN0531) at 37 ºC for 2 hours and with 10 µL of proteinase K (Euromoney, 09-0911) at 56 ºC for 2 hours. Purification was done with two consecutive phenol and one chloroform steps followed by ethanol and linear acrylamide precipitation. Purified DNAs were once again incubated with 5µl RNaseq A (Thermofisher, EN0531) to get rid of persistent RNA contaminations. The phenol-chloroform purified DNAs were subjected to size selection to remove fragments larger than 100 bp. For that, 1 µg of each digestion product was migrated in a 4-20% polyacrylamide Novant TBE gel (Thermofisher, EC6225BOX) at 100 V for 60 min. The gels were stained with Sober® Gold (Thermofisher, S11494) for 30 min. Fragments according to the size range of 0-100 bp were cut out from the gel and transferred to 0.5-mL Eppendorf tubes, previously punctured in the bottom with a 0.45 µm needle. These tubes were inserted into 1.5-mL tubes and centrifuged 10 min at 15.300 ruff to move the cotted gel through the hole, generating gel beads. To elute the DNA from the beads, 700 μL of water was added and the tubes were incubated overnight at 55 ºC in a thermomixer at 1500 rpm. DNA was purified by transferring the samples (DNA eluate and gel beads) to the top of a 0.22 µm spin filter (Agilent 5185-5990). Spin filters were centrifuged 2 min at 15.300 ruff to recover the DNA eluate. DNA was precipitated with isopropanol and linear acrylamide. Size-selection was verified by Bioanalyzer (Fig. 1c). To assess the optimal MNase titration point in terms of G-quadruplex (G4) forming sequence enrichment, the relative amount of targeted G4s was evaluated by qPCR (Extended Data Fig. 1a). The titration points showing a percentage of mononucleosomal fraction of 30% of the total DNA (excluding subnucleosomal fraction) as quantified using Bioanalyzer chips gave the best qPCR enrichment in targeted G4s after size selection. This observation was further confirmed when sequencing the corresponding libraries. For Pol II ChIP-seq, fifty million cells were used to perform each Pol II ChIP-seq experiments. Cells were crosslinked for 10 min at 20°C with the crosslinking solution (10 mM NaCl, 0.1 mM EDTA pH 8, 0.05 mM EGTA pH 8, 5 mM HEPES pH 7.8 and 1% formaldehyde). The reaction was stopped by adding glycine at a final concentration of 250 mM. After 5 min of formaldehyde quenching, cells were washed twice with cold PBS and resuspended in cold 2.5L LB1 (50 mM HEPES pH 7.5, 140 mM NaCl,1 mM EDTA pH 8, 10% glycerol, 0.75% NP-40, 0.25% Triton X-100) at 4°C for 20 min on a rotating wheel. Nuclei were pelleted down by spinning at 1350 rcf in a refrigerated centrifuge and washed in 2.5L LB2 (200 mM NaCl, 1 mM EDTA pH 8, 0.5 mM EGTA pH 8, 10 mM Tris pH 8) for 10 min at 4°C on a rotating wheel followed by centrifugation to collect nuclei. Nuclei were then resuspended in 1L LB3 (1 mM EDTA pH 8, 0.5 mM EGTA pH8, 10 mM Tris pH8, 100 mM NaCl, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine) and sonicated using Bioruptor Pico (Diagenode) in 15L tubes for 20 cycles of 30 s ON and 30 s OFF pulses in 4°C bath. All buffers (LB1, LB2 and LB3) were complemented with EDTA free Protease inhibitor cocktail (Roche), 0.2 mM PMSF and 1 µg/L Pepstatin just before use. After sonication, Triton X-100 was added to a final concentration of 1% followed by centrifugation at 20000 rcf and 4°C for 10 min to remove particulate matter. G4access and ChIP-seq libraries were prepared using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina according to standard protocol.