After four days of OHT treatment, cells were collected and counted. Cells were washed twice in cold DPBS and resuspended in full media. Samples were crosslinked for 10 minutes rotating at room temperature in 1% formaldehyde (106 cells/ml). Crosslinking was stopped by adding glycine at the final concentration of 0.2 M for 5 minutes at RT. Samples were washed twice in cold DPBS and pellets were snap frozen. 2-5 × 106 cells per sample were lysed in 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% IGEPAL (Sigma, I3021) supplemented with protease inhibitor (Thermo Scientific, 78430) for 15 minutes on ice. Nuclei were washed twice in cold lysis buffer, resuspended in 50 μl of NEBuffer 3.1 (NEB, B7003S) supplemented with 0.3% SDS and incubated at 62°C for 10 minutes. After diluting and quenching the SDS by adding 57.5 μl of NEBuffer 3 and 12.5 μl of 20% Triton X-100 (Sigma, 93443), samples were incubated at 37°C for 60 minutes. Nuclei were spun and incubated in 250 μl of 1× DpnII buffer with 600 U of DpnII restriction enzyme (NEB, R0543L) overnight at 37°C. An additional 200 U of DpnII were added to each sample the following day and incubated for a further 2 hours.  After heat inactivation of DpnII for 20 min at 65°C, restriction fragments ends were filled by DNA polymerase I, Large (Klenow) Fragment (NEB, M0210), 0.8 U/μl of DNA, in 50 μl of 0.3 mM biotin-14-dATP (Thermo Fisher, 19524016), 0.3 mM dCTP/ dGTP/ dTTP for 1.5 hours at 37°C.  900 μl of ligation master mix were then add to the samples: 1.33x NEB T4 DNA ligase buffer (NEB, B0202), 1.1% Triton X-100, 1.33 mg of Bovine Serum Albumin and 2000 U of T4 DNA Ligase (NEB, M0202). Samples were incubated for 4 hours rotating. Nuclei were spun and resuspended in water and digested with proteinase K (Sigma, P6556) 1.2 mg/ml and 0.8% SDS at 55°C for 30 minutes. 130 μl of 5 M NaCl were added and samples were incubated at 65°C overnight. After ethanol precipitation, DNA was resuspended in 500 μl of 10 mM Tris-HCl pH8.0 and washed trice using the same buffer using Amicon filters (Millipore, UFC503096). Samples were diluted in 50 mM Tris pH 8.0, 0.1% SDS, 10 mM EDTA and sonicated to about 500 bp using a probe-based sonicator. Washes were then repeated as in the previous step. Biotinylated DNA was pulled down using 30 l of 10 mg/ml Dynabeads MyOne Streptavidin T1 beads (Life technologies, 65602) in 10 mM Tris-HCl (pH 7.5); 1 mM EDTA; 2 M NaCl for 15 minutes at room temperature rotating. Beads were washed in TWB: 5 mM Tris-HCl (pH 7.5); 0.5 mM EDTA; 1 M NaCl; 0.05% Tween 20 for 2 minutes at 55° C twice, resuspended in 20 l 1× NEB T4 DNA ligase buffer, transferred to a new tube and incubated for 30 minutes at room temperature with 100 μl of: 1x NEB T4 DNA ligase buffer, 0.5 mM dNTPs, 50 U T4 PNK (NEB, M0201), 12 U T4 DNA polymerase I (NEB, M0203), 5 U DNA polymerase I, Large (Klenow) Fragment. After two washes in TWB at 55°C for 2 minutes, beads were transferred to 1× NEBuffer 2 (NEB, B7002S) and moved to a new tube. Beads were then incubated at 37°C for 30 minutes in 100 l of 0.9X NEBuffer 2, 0.5 mM dATP and 25 U Klenow exo minus (NEB, M0212). Beads were washed again as before and transferred to T4 ligation buffer. DNA was ligated for 2.5 hours with Illumina adaptors in 50 μl of the following reaction mix: 1X T4 ligation buffer, 1.2 µM Illumina adaptors, 1U T4 DNA ligase. Beads were washed once again with TWB as before, to be then resuspended and stored in 50 μl of 10 mM Tris-HCl (pH8.0).  Libraries were amplified in six parallel 50 µl PCR reactions using Illumina primers and 4 μl of beads (or DNA removed off the beads by heating at 95°C for 20 min) per reaction after testing optimal amplification cycles. Size selection was performed on an agarose gel, isolating fragments between 300-700 base pairs, followed by purification with QIAquick Gel Extraction Kit (Qiagen 28704). Each library was test sequenced at low depth to test its quality, and then selected libraries were sequenced on the equivalent of approximately one mid-output lane of Illumina NextSeq 500/550 (75 bp  paired end reads). All data were used in the final analyses.
GEO Accession
GSM4458502