GSM3771270: CTCFAID_CTCF_I_minusIAA_PFA_2; Mus musculus; ChIP-Seq
ChIP-seq/Mnase-seq: For interphase, 2.5x106 fixed cells (1% formaldehyde for 10min followed by 5min with 0.125 M Glycine) were resuspended in 2ml swelling buffer (25mM Hepes pH 7.95, 10mM KCl, 10mM EDTA; freshly supplemented with 1× protease inhibitor cocktail – 04 693 116 001 PIC-Roche, and 0.5% IGEPAL), incubated for 30min on ice, and passed 40 times in a dounce homogenizer to recover nuclei. For mitotic cells, the homogenization step was omitted. Mitotic and interphase cells were then treated in parallel to sonicate the chromatin in 300μl of TSE150 (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl pH8, 150mM NaCl; freshly supplemented with 1× PIC) using 1.5ml tubes (Diagenode) and a Bioruptor Pico (Diagenode; 7 cycles divided into 30 s ON–30 s OFF sub-cycles at maximum power, in circulating ice-cold water). After centrifugation (30min, full speed, 4 °C), the supernatant was pre-cleared for 2h with 50μl of protein G Sepharose beads (P3296-5 ML Sigma) 50% slurry, previously blocked with BSA (500 μg/ml; 5931665103 Roche) and yeast tRNA (1 μg/ml; AM7119 Invitrogen). 20μl were set apart (input) before over-night immunoprecipitation at 4°C on a rotating wheel with 4µl of anti-CTCF (Active Motif 61311) or 4µl of anti-SMC1 (Bethyl Laboratories A300-055A) antibodies in 500μl of TSE150. Protein G beads (25μL 50% slurry) were added for 4 h rotating on-wheel at 4 °C. Beads were pelleted and washed for 15min rotating on-wheel at 4 °C with 1ml of the following buffers: 3 washes with TSE150, 1 wash with TSE500 (as TSE150 but 500 mM NaCl), 1 wash with Washing buffer (10mM Tris-HCl pH8, 0.25M LiCl, 0.5% NP-40, 0.5% Na-deoxycholate, 1mM EDTA), and 2 washes with TE (10mM Tris-HCl pH8, 1mM EDTA). Elution was performed in 100μl of elution buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH 8) for 15min at 65 °C after vigorous vortexing. Eluates were collected after centrifugation and beads rinsed in 150μl of TE-SDS1%. After centrifugation, the supernatant was pooled with the corresponding first eluate. For both immunoprecipitated and input chromatin, the crosslinking was reversed overnight at 65 °C, followed by proteinase K treatment, phenol/chloroform extraction and ethanol precipitation. H3 ChIP-seq was performed on MNase-digested chromatin using 2.5x106 cells fixed with formaldehyde as described above. Fixed cells were resuspended in 500µl of MNase buffer (50 mM Tris-HCl pH8, 1 mM CaCl2, 0.2% Triton X-100) supplemented with protease inhibitor cocktail (PIC; 04 693 116 001 Roche). Cells were pre-incubated for 10min at 37°C with 16U of MNase (Expressed in KUntiz, 1 Kunitz is equivalent to 10 gel units, NEB M0247S) added to the reaction. Cells were incubated for further 10min at 37°C, inverting the tubes occasionally. The reaction was stopped on ice by adding 500μl of 2×STOP buffer (2% Triton X-100, 0.2% SDS, 300mM NaCl, 10mM EDTA). Tubes were left rotating overnight on a wheel to allow diffusion of the digested fragments. The cell suspension was spun down and the supernatant stored at -80°C. Subsequently, 50µl of chromatin were brought to a final volume of 500µl with TSE150 (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl pH8, 150mM NaCl), freshly supplemented with protease inhibitor cocktail (PIC-Roche, 04 693 116 001), and ChIP was performed as described above using 5µg of anti-Histone H3 rabbit polyclonal (Abcam; ab1791).