This was followed by two washes in 50 mM EDTA incubated at 50 ☌ for 3 min, two washes in Tween Wash buffer incubated at 55 ☌ for 2 min, and one wash in 10 mM Tris-HCl pH 7.5. Beads were resuspended in 50 mM EDTA and incubated at 50 ☌ for 30 min to quench the transposase reaction. For tagmentation, beads were washed twice in 100 μl of freshly prepared tagmentation reaction buffer (10 mM Tris–HCl, pH 8.0, 5 mM MgCl2, 10% dimethylformamide), and resuspended in 25 μl of the tagmentation reaction buffer and 1 μl Tagment DNA Enzyme from the Nextera DNA Sample Prep Kit (Illumina) and incubated at 55 ☌ for 10 min in a thermocycler with interval shaking. Samples were then washed twice in TWB with 2 min incubation at 55 ☌ shaking. To enrich for ligation events, 5 μl of Streptavidin C-1 beads were washed in Tween Wash Buffer (TWB, 5 mM Tris–HCl pH 7.5, 0.5 mM EDTA pH 8.0, 1M NaCl, 0.05% Tween-20), resuspended in 10 μl of 2X biotin binding buffer (10 mM Tris–HCl pH 7.5, 1 mM EDTA pH 8.0, 2 M NaCl), added to 10 μl of the samples and incubated at room temperature for 15 min with rotation. Of note, we obtained between 3 and 8 ng of DNA for CTCF HiChIP from 15 million cells. DNA was purified on kept a column with the Qiagen mini Elute kit, eluted in 12 μl of H2O, and quantified using Qubit. Chromatin was eluted and decrosslinked by adding 100 μl of freshly prepared elution buffer (0.5% SDS, 300 mM NaCl, 5 mM EDTA pH 8.0, 10 mM Tris–HCl pH 8.0) and 10 μl of proteinase K at 10 mg/ml for 45 min at 55 ☌ and at least 1.5 h at 67 ☌. Briefly, beads were washed twice with 500 μl cold Low-salt wash buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 2 mM EDTA pH 8.0 NaOH, 0.1% SDS, 1% triton X-100), twice with 500 μl cold high-salt wash buffer (20 mM Tris–HCl pH 7.5, 500 mM NaCl, 2 mM EDTA pH 8.0 NaOH, 0.1% SDS, 1% triton X-100), and twice with 500 μl cold LiCl-containing wash buffer (10 mM Tris–HCl pH 8.0, 250 mM LiCl, 1 mM EDTA pH 8.0 NaOH, 1% NP-40, 1% sodium deoxycholate). Immunoprecipitation was performed by overnight incubation rotating in cold room and washes were performed. CTCF antibody (5 μl, Millipore 07-729) was combined with 50 μl of protein-A magnetic beads (Dynabeads) for one hour at room temperature, and added to sonicated chromatin from 15 million cells. Samples were sonicated on the bioruptor for 15 min (an agarose gel was performed to make sure that the sonicated DNA smear was in the range of 250–600 bp), supplemented with 1% Triton X-100 and centrifuged 5 min at 16,000 rcf at 4 ☌. Following ligation, nuclei were pelleted and resuspended in 350 μl cold Nuclei Lysis Buffer (50 mM Tris–HCl pH 7.5, 10 mM EDTA, 1% SDS, and 1x Protease Inhibitors) with incubation rotating in the cold room for 10 min. Ligation mix was added to the samples (150 μl 10x ligation buffer (NEB B0202S), 7.5 μl 20mg per ml BSA (NEB B9001S), 150 μl Triton X-100 10%, 4000 units T4 DNA ligase (NEB M0202S), and 655.5 μl H2O) for 4 h at RT with rotation. To fill in the restriction fragment overhangs and mark the DNA ends with biotin, 1.5 μl 10 mM dCTP, 1.5 μl 10 mM dGTP, 1.5 μl 10 mM dTTP, 37.5 μl 0.4 mM biotin-14-dATP (Life Technologies 19524-016), and 50 units Klenow (DNA polymerase I large fragment, NEB M0210L) were added to each tube, and incubated for 60 min at 37 ☌. MboI was inactivated by incubating the samples 20 min at 62 ☌. Chromatin was then digested by adding 50 μl of NEBuffer 2 10X and 350 units of MboI (NEB R0147M) at 37 ☌ for 2 h while rotating at 950 rpm. SDS was quenched by adding 285 μl of H2O and 50 μl of Triton X-100 10%, and incubating at 37 ☌ for 15 min. Cell pellets were collected, washed once in 500 μl ice-cold lysis buffer and then incubated in 100 μl 0.5% SDS at 62 ☌ for 10 min. Cells were then lysed in 500 μl ice-cold lysis buffer (10 mM Tris–HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA-630, protease inhibitor cocktail (Roche complete, EDTA-free)) rotating at 4 ☌ for 30 min. HiChIP was performed with 15 million cells, as per the original protocol54. Pellets were washed twice with ice-cold PBS, snap-frozen in liquid nitrogen and stored at −80 ☌. Cells were fixed in culture medium with 1% formaldehyde at RT for 10 min and quenched with 0.125 M glycine. HiChIP was performed in triplicate (for NSD2 High: one KMS11 and two NTKO replicates from independent cultures, for NSD2 Low, two replicates from one clone and one replicate from the other one, from independent cultures).
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