In situ contact libraries were generated according to the in situ Hi-C published protocol5 (link) through the proximity ligation with modifications. In brief, up to 15 million crosslinked cells were resuspended in 500 μL of ice-cold Hi-C lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.2% NP-40, 1X Roche protease inhibitors - 11697498001) and rotated at 4° C for 30 minutes. For cell amounts greater than 15 million, the cell pellet was split in half for contact generation and then recombined for the sonication. Nuclei were pelleted at 4° C for 5 minutes at 2500 rcf and the supernatant was discarded. Pelleted nuclei were washed once with 500 μL of ice-cold Hi-C lysis buffer. Supernatant was removed again and pellet was resuspended in 100 μL of 0.5% SDS and incubated at 62° C for 10 minutes with no shaking or rotation. 285 μL of water and 50 μL of 10% Triton X-100 were added and samples were rotated at 37° C for 15 minutes to quench the SDS. 50 μL of NEB Buffer 2 and 15 μL of 25 U/μL MboI restriction enzyme (NEB, R0147) were then added and sample was rotated at 37° C for 2 hours. For lower starting material less restriction enzyme was used: 15 μL was used for 10–15 million cells, 8 μL for 5 million cells, and 4 μL for 1 million cells. MboI was then heat inactivated at 62° C for 20 minutes with no shaking or rotation. To fill in the restriction fragment overhangs and mark the DNA ends with biotin, 52 μL of incorporation master mix was then added: 37.5 μL of 0.4 mM biotin-dATP (Thermo Fisher 19524016), 4.5 μL of a dCTP, dGTP, and dTTP mix at 10 mM each, and 10 μL of 5 U/μL DNA Polymerase I, Large (Klenow) Fragment (NEB, M0210). The reactions were then rotated at 37° C for 1 hour. 948 μL of ligation master mix was then added: 150 μL of 10X NEB T4 DNA ligase buffer with 10 mM ATP (NEB, B0202), 125 μL of 10% Triton X-100, 3 μL of 50 mg/mL BSA (Thermo Fisher AM2616), 10 μL of 400 U/μL T4 DNA Ligase (NEB, M0202), and 660 μL of water. The reactions were then rotated at room temperature for 4 hours. After proximity ligation, the nuclei with in-situ generated contacts were pelleted at 2500 rcf for 5 minutes at room temperature and the supernatant was removed.