For RNA, primers were designed to amplify desired genomic regions that correspond to peaks of boundary RNA (GRO-seq) at the B1 and B2 CTCF sites of the INK4a/ARF TAD boundary. The amplified products were cloned into the pcDNA3 BoxB plasmid (a gift from Howard Chang, Addgene 29729). All clones were confirmed by Sanger sequencing and subsequently used for RNA synthesis using T7 RNA polymerase (Promega P207e) and biotin RNA labeling mix (Roche 11685597910). Primer sequences are listed in Supplemental Table S1 .
RNA immunoprecipitation was adopted from Jayani et al. (2017) (link), with some modifications. HeLa cells were grown to 90%–95% confluency and were harvested by scraping and washed twice with ice-cold 1× PBS to gently resuspend in 2 mL of PBS. A volume of 2 mL of nuclear isolation buffer (NIB; 40 mM Tris–HCl at pH 7.5, 20 mM MgCl2, 1.28 M sucrose, 4% Triton X-100, 1 mM PMSF, protease inhibitors, and 20 U/mL SUPERase inhibitor; Thermo Fisher Scientific AM2694) was added, and the pellet was gently resuspended. Then, 6 mL of distilled water was added and kept on ice for 20 min with intermittent gentle shaking. Nuclei were pelleted by centrifugation at 2500g for 5 min at 4°C. The pellet was resuspended in 1 mL of RIP buffer (25 mM Tri–HCl at pH 7.4, 150 mM KCl, 0.5 mM DTT, 0.5% NP-40, 1 mM PMSF, protease inhibitors, and 20 U/mL SUPERase inhibitor) and incubated on ice for 5 min. Nuclei were sheared by 10 cycles of sonication (30 sec on and 30 sec off) in a Bioruptor (Diagenode) followed by centrifugation at 12,000 rpm for 10 min at 4°C. One microgram of biotinylated RNA was incubated with 20 μL of RNA structure buffer (RSB; 10 mM Tris–HCl at pH 7.0, 100 mM KCl, 10 mM MgCl2, 1 mM PMSF, protease inhibitors, and 20 U/mL SUPERase inhibitor) for 5 min at room temperature. Folded RNA was mixed with 1 mg of nuclear extract in 500 µL of RIP buffer and rotated for 1 h at 4°C. Fifteen microliters of Dynabeads MyOne streptavidin beads were added, and rotation continued for one more hour. Samples were washed three times with RIP buffer, and beads with proteins were boiled in 2×SDS for 10 min. Immunoblotting for CTCF was performed using CTCF antibody (Cell Signaling Technology 3418).
RNA immunoprecipitation was adopted from Jayani et al. (2017) (link), with some modifications. HeLa cells were grown to 90%–95% confluency and were harvested by scraping and washed twice with ice-cold 1× PBS to gently resuspend in 2 mL of PBS. A volume of 2 mL of nuclear isolation buffer (NIB; 40 mM Tris–HCl at pH 7.5, 20 mM MgCl2, 1.28 M sucrose, 4% Triton X-100, 1 mM PMSF, protease inhibitors, and 20 U/mL SUPERase inhibitor; Thermo Fisher Scientific AM2694) was added, and the pellet was gently resuspended. Then, 6 mL of distilled water was added and kept on ice for 20 min with intermittent gentle shaking. Nuclei were pelleted by centrifugation at 2500g for 5 min at 4°C. The pellet was resuspended in 1 mL of RIP buffer (25 mM Tri–HCl at pH 7.4, 150 mM KCl, 0.5 mM DTT, 0.5% NP-40, 1 mM PMSF, protease inhibitors, and 20 U/mL SUPERase inhibitor) and incubated on ice for 5 min. Nuclei were sheared by 10 cycles of sonication (30 sec on and 30 sec off) in a Bioruptor (Diagenode) followed by centrifugation at 12,000 rpm for 10 min at 4°C. One microgram of biotinylated RNA was incubated with 20 μL of RNA structure buffer (RSB; 10 mM Tris–HCl at pH 7.0, 100 mM KCl, 10 mM MgCl2, 1 mM PMSF, protease inhibitors, and 20 U/mL SUPERase inhibitor) for 5 min at room temperature. Folded RNA was mixed with 1 mg of nuclear extract in 500 µL of RIP buffer and rotated for 1 h at 4°C. Fifteen microliters of Dynabeads MyOne streptavidin beads were added, and rotation continued for one more hour. Samples were washed three times with RIP buffer, and beads with proteins were boiled in 2×SDS for 10 min. Immunoblotting for CTCF was performed using CTCF antibody (Cell Signaling Technology 3418).
