Anti ctcf

UV crosslinking RIP was carried out as described before^{33 (link)}. Two 10 cm² dishes of HT29 cells with 90%-100% confluence were washed twice with 5 ml cold PBS and irradiated at 300 mJ/cm² at 254 nm in a Stratalinker. Cells were collected and resuspended in 1 ml RIP buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM PMSF, 2 mM VRC, protease inhibitor cocktail). Then cells were homogenized by 3 rounds of sonication on ice. Insoluble material was removed by centrifugation and the supernatant was pre-cleared with Dynabeads G (Invitrogen) with 20 μg/ml yeast tRNA at 4 °C for 30 min. The pre-cleared lysate was incubated with Dynabeads G that were pre-coated with 2 μg antibodies of anti-CTCF (Millipore), anti-TCF4 (Millipore) or IgG (Sigma) for 4 h at 4 °C. The beads were washed sequentially with washing buffer I (50 mM Tris-HCl, pH 7.5, 1 M NaCl, 1% NP-40, 1% sodium deoxycholate, 2 mM VRC) and washing buffer II (50 mM Tris-HCl, pH 7.5, 1 M NaCl, 1% NP-40, 1% sodium deoxycholate, 2 mM VRC, 1 M urea) for multiple times. The immunoprecipitated complex was eluted from Dynabeads G by adding 140 μl elution buffer (100 mM Tris-HCl, pH 7.0, 5 mM EDTA, 10 mM DTT, 1% SDS). 5 μl of 10 mg/ml proteinase K was then added to the retrieved RNA samples and incubated at 55 °C for 30 min, followed by RNA extraction and qPCR.

Biotin-labeled RNAs pull-down assay was performed as described^{33 (link),57 (link),58 (link)} with minor modifications. Biotin-labeled CCAT1-L truncation probes were in vitro transcribed with the Biotin RNA Labeling Mix (Roche) and AmpliScripe T7/SP6-flash Transcription Kit (Epicentre). 4 μg biotinylated RNA was denatured for 5 min at 65 °C in PA buffer (10 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 100 mM NH₄Cl) and slowly cooled down to RT. 2 × 10⁷ HT29 cell pellet was used for each assay. Briefly, HT29 cell nuclei^{33 (link)} were resuspended in 1 ml RIP buffer (25 mM Tris-HCl, pH 7.5, 150 mM KCl, 0.5 mM DTT, 0.5% NP-40, 1 mM PMSF, 2 mM VRC, protease inhibitor cocktail). The nuclei were sonicated on ice followed by centrifugation at 13 000 rpm for 10 min at 4 °C. The supernatant was transferred to a new tube and pre-cleared by applying 40 μl Streptavidin Dynabeads for 20 min at 4 °C. Then 20 μg/ml yeast tRNA was added to block nonspecific binding and incubated for 20 min at 4 °C. Folded RNAs were then added and incubated for 1.5 h at RT, followed by the addition of 40 μl Streptavidin Dynabeads to incubate for 1.5 h. Beads were washed with RIP buffer containing 0.5% sodium deoxycholate and denatured in 1× SDS loading buffer. The retrieved proteins were analyzed by western blot with anti-CTCF (Millipore) and anti-TCF4 (Millipore) antibodies.