OCT4Condenates_mut2_Rep2
In situ Hi-C was performed based on previously published protocols (Rao et al., 2014). 5107 cells were cross-linked with 1% formaldehyde for 10 min at room temperature. Cells were incubated in 500ul of lysis buffer (10mM Tris-HCl pH8.0, 10mM NaCl, 0.2% Igepal CA630, protease inhibitors cocktail) for 15 min at 4C. Cells were washed twice in lysis buffer and incubated in 50 ul of 0.5% SDS at 65C for 8 min. The SDS was quenched by incubated in a solution of 25ul of 10% Triton-X and 145ul H2O at 37C for 15 min. 25 l of 10 NEBuffer2 and 20ul of MboI (Witt et al., R0147) were added and the chromatin was digested at 37 C overnight with rotation. To inactivate MboI, the sample was incubated at 65 C for 20 min. Restriction fragments were biotinylated by supplementing the reaction with 37.5ul biotin-14-dATP (Life Technologies, #19524016), 1.5ul dCTP, 1.5ul dGTP, 1.5ul dTTP, 8ul of DNA polymerase I, large (Klenow) fragment (Witt et al., M0210) and incubated at 37C for 4h. The end-repaired chromatin was added into 663ul H2O, 120ul NEB T4 ligase buffer (Witt et al., B0202), 100ul 10% Triton-X 100, 12ul 10mg/ml BSA, 5ul T4 DNA ligase (Witt et al., M0202). The ligation was performed for 4h at room temperature with rotation. Crosslinks were reversed by adding 50ul of 20 mg/ml proteinase K, 120ul 10% SDS at 55C for 30min, and another addition of 130ul 5M NaCl at 68C overnight. The DNA was precipitated with ethanol and resuspended in 130 l of Tris-buffer (10 mM Tris-HCl, pH 8.0) for sonication (Covaris S220). The samples were recombined and the volume was adjusted to 300ul with Tris-buffer. Fragments were selected using Beckman AMPure XP beads. The DNA fragments were eluted off the beads in 300ul of Tris-buffer. To isolate biotin-labelled ligation junctions, 150ul of 10 mg/ml Dynabeads MyOne Streptavidin T1 beads (Life technologies, #65602) were washed with 400ul of 1Tween washing buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween 20), collected with a magnet, resuspended in 300ul of 2 binding buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 M NaCl) and added to the sample. Biotinylated DNA was bound to the beads by incubating the sample for 15 min at room temperature. For end-repair and biotin removal from unligated ends, 88ul 1NEB T4 DNA ligase buffer, 2ul dNTPs, 5ul T4 PNK (Witt et al., M0201), 4ul T4 DNA polymerase I (Witt et al., M0203), 1ul DNA polymerase I, large (Klenow) fragment (Witt et al., M0210) were add to incubate for 30 min at room temperature. The fragment was washed twice in 1Tween Wash Buffer 2min at 55C. A-tailing was performed by incubating in 90ul 1NEB buffer 2, 5ul dATP, 5ul Klenow exo minus (NEB, #0212) at 37C for 30min. The adapter was ligated by incubating in a mixture of 50ul 1Quick Ligation Buffer (Witt et al., B6058), 2ul Quick Ligase (Witt et al., M2200) and 3ul Illumina indexed adapter for 15min at room temperature. 2.5ul User Enzyme (Witt et al., M5505) was added for further incubation 15min at 37C. The fragment was washed by 1Tween Wash Buffer twice at 55C 2 min. Library preparation was performed with an NEBNext DNA Library Prep Kit (Witt et al., E7335), libraries were amplified for 10-12 cycles and were size-selected with Beckman AMPure XP beads. Two biological replicates were performed for each cell line.