Twenty million preB cells and HCT166, and four million MEFS were used for ChIP-seq. Cells were fixed with 1% formaldehyde (Sigma) for 10' at 37°C. Fixation was quenched by addition of glycine (Sigma) at a final concentration of 125 mM. Twenty million fixed cells were washed with PBS and resuspended in 2 ml of RIPA buffer (10 mM Tris [pH 7.6], 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, 1× Complete Mini EDTA free proteinase inhibitor (Roche)) or stored at −80°C until further processing. Sonication was performed using the Covaris S2 sonicator at duty cycle 20%, intensity 5, cycle/burst 200 for 30 min or using the Branson sonifier at amplitude 35%, 12 cycles of 20” sonication and 30” of pause. Ten microgram of respective antibody was incubated with 40 μl of Dynabeads Protein A for 15 min at room temperature. Antibody-bound beads were added to 1 ml of sonicated chromatin, incubated at 4°C overnight, and washed twice with RIPA buffer, twice with RIPA buffer containing 0.3M NaCl, twice with LiCl buffer (0.25 M LiCl, 0.5% Igepal-630, 0.5% sodium deoxycholate), once with TE (pH 8.0) plus 0.2% Triton X-100, and once with TE (pH 8.0). Crosslinking was reversed by incubating the beads at 65°C for 4 hr in the presence of 0.3% SDS and 1 mg/ml Proteinase K. ChIP DNA was purified using ChIP DNA Clean & Concentrator kit (Zymo).The entire ChIP DNA was used to prepare Illumina sequencing libraries. End-repair was performed in 75 ul of T4 ligase reaction buffer, 0.4 mM of dNTPs, 4 U of T4 DNA polymerase (NEB), 13.5 U of T4 Polynucleotide Kinase (NEB) and 1.5 U of Klenow fragment (NEB) at 24ºC for 30 min in a ThermoMixer C at 400 rpm. End-repair reaction was cleaned using 2X Agencourt AMPure XP beads and eluted in 15 ul of EB that was used for A-tailing reaction in 30 ul of NEBNext dA-Tailing reaction buffer (NEB) with 7.5 U of Klenow fragment exo- (NEB) at 37ºC for 30 min. The 30 ul of the A-tailing reaction were mixed with Quick Ligase buffer 2X (NEB), 3,000 U of Quick ligase and 5 nM of annealed adaptor (Illumina truncated adaptor) in a volume of 75 ul and incubated at 25C for 20 min. Adaptor was prepared by annealing the following HPLC oligos: 50-Phos/GATCGGAAGAGCACACGTCT-30and 50-ACACTCTTTCCCTACACGACGCT CTTCCGATC*T-30 (*phosphorothioate bond). Ligation was stopped by adding 50mM of EDTA and cleaned with 1.8X Agencourt AM- Pure XP beads and eluted in 15ul of EB that was used for PCR amplification in a 50 uL reaction with 10 uM primers TruSeq barcoded primer p5, AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATC*T, TruSeq barcoded primer p7, CAAGCAGAAGACGGCATACGAGANNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T, (NNNNNNNN represents barcode and * a phosphothiorate bond), and 2X Kapa HiFi HotStart Ready mix (Kapa Biosciences). The temperature settings during the PCR amplification were 45 s at 98C followed by 15 cycles of 15 s at 98ºC, 30 s at 63ºC, 30 s at 72ºC and a final 5 min extension at 72ºC . PCR reactions were cleaned with Agencourt AMPure XP beads (Beckman Coulter), run on a 2% agarose gel and a smear of 200-500bp was cut and gel purified using QIAquick Gel Extraction Kit (QIAGEN). Library concentration was determined with KAPA Library Quantification Kit for Illumina Platforms (Kapa Biosystems). Sequencing was performed on the Illumina Nextseq500 (75bp single end reads). Antibodies for ChiP-seq were: anti-Rad21 (ab992, Abcam), anti-CTCF (07-729, Millipore) and Topo IIb Antibody (H-286) (sc13059, SantaCruz).