GSM7993396: Δ3XCTCF_ESC_Gbx2SE_rep1; Mus musculus; OTHER
Capture-C experiments were performed as previously described. 5 _ 106 cells were crosslinked with 2% formaldehyde for 10 minutes and quenched with 0.125 M glycine for 10 minutes. Cells were washed with PBS and resuspended in lysis buffer (10 mM Tris pH 8, 10 mM NaCl, 0.2% NP-40 and 1_ protease inhibitors) during 20 minutes on ice. Following centrifugation, the pellet was resuspended in 215 µL 1_CutSmart buffer and transferred to a microcentrifuge tube. The resuspended pellet was mixed with 60 ul 10_ CutSmart buffer, 393,5 µl water and 9,5 µl 20% (vol/vol) SDS (0.28% final concentration) (Invitrogen, cat. no. AM9820) followed by 1 h incubation at 37¡C while shaking on a thermomixer at 500 rpm (intermittent shaking_ 30s on / 30s off).  Then, 20% vol/vol Triton X 100 was added at a final concentration of 1.67% vol/vol followed by another incubation at 37¡C for 1h while shaking. Chromatin was digested by adding 25 µL NlaIII (250 U, R0125L) and incubating at 37 ¡C for several hours, followed by addition of another 25 µL of NlaIII and incubation at 37 ¡C overnight. The digested chromatin was ligated with 8 µl (240U) of T4 DNA ligase (Life Tech, cat. no. EL0013) for 18 hours at 16¡C. Samples were treated with Proteinase K (3U; Thermo Fisher, cat. no. EO0491) and RNase A (7,5 mU; Roche_ 1119915), and DNA was purified using the Qiagen kit (28506). After checking the quality of the digestion and subsequent ligation, chromatin was sonicated for 30 cycles (30 s on, 30 s off, 25% amplitude) using an EpiShear probe sonicator (Active Motif) and DNA samples were purified using AMPure XP SPRI beads (Beckman Coulter, cat. no. A63881). Libraries were prepared using the NEBNext Ultra II kit (New England Biolabs, cat. no. E7645S/L). Index primers set 1 and 2 from the NEBNext Multiplex Oligos for Illumina kit (New England Biolabs, E7335S/L E7500S/L) were incorporated using Herculase II Fusion Polymerase Kit (Agilent, cat. no. 600677). The resulting libraries were pooled (six libraries/pool) and a double Hybridization capture using ssDNA probes was performed following a modified version of the Roche HyperCapture streptavidin pull-down protocol (Roche, cat. no. 09075763001) described in 81. Libraries were sequenced using Novaseq6000_150PE_2,25Gb/lib (15 Mreads/lib). For each of the investigated cell lines, Capture-C experiments were performed as two independent biological replicates.