Proteinase k solution

The cells were seeded in a 24-well falcon dish at a concentration of 2×10⁵ cells/mL and incubated for 24 h after the addition of each reagent. Reagent-treated cells were centrifuged at 300×g for 5 min and washed once with PBS. The cell pellet was suspended in 100 μL of cell lysis buffer (10 mM Tris–HCl buffer, pH 7.4, containing 10 mM EDTA and 0.5% Triton X-100) and kept at 4°C for 10 min. The cell lysate was centrifuged at 16,000×g for 20 min. The supernatant (100 μL) was incubated with 2 μL of RNase A (20 mg/mL; Macherey-Nagel, Düren, Germany) at 37°C for 60 min, and then with 2 μL of proteinase K solution (20 mg/mL; Wako, Japan) at 37°C for 60 min. After adding 20 μL of 5 M NaCl and 120 μL of isopropyl alcohol, the mixture was kept at –30°C overnight. The precipitate was then collected by centrifugation at 16,000×g for 15 min and washed twice with 70% ethanol. After the removal of ethanol, samples were allowed to stand for 5 min on a clean bench to volatilize the remaining ethanol. Fragmented DNA was then dissolved in TE buffer (10 mM Tris–HCl, pH 7.4, and 1 mM EDTA) and subjected to 2% agarose gel electrophoresis at 100 V for 45 min. Fragmented DNA was stained with 0.5 μg/mL ethidium bromide solution (Genesee Scientific, San Diego, USA). The electrophoresed gel was photographed using Printgraph CMOS I (ATTO Co., Tokyo, Japan).

Cells were centrifuged at 300×g for 5 min and washed once with PBS. The cell pellet was suspended in 100 μL of cell lysis buffer (10 mM Tris–HCl buffer, pH 7.4 containing 10 mM EDTA and 0.5% Triton X-100), and kept at 4°C for 10 min. Cell lysate was centrifuged at 16,000×g for 20 min. The supernatants (100 μL) were incubated with 2 μL of RNase A (20 mg/mL; MACHEREY-NAGEL, USA) at 37°C for 60 min, and then with 2 μL of proteinase K solution (20 mg/mL; Wako, Japan) at 37°C for 60 min. After adding 20 μL of 5 M NaCl and 120 μL of isopropyl alcohol, these mixtures were kept at −30°C overnight. The precipitate was then collected by centrifugation at 16,000×g for 15 min and washed twice with 70% ethanol. After removal of ethanol, samples were allowed to stand for 5 min on a clean bench to volatilize the remaining ethanol. DNA samples were then dissolved in TE buffer (10 mM Tris–HCl, pH 7.4 and 1 mM EDTA), and subjected to 2% agarose gel electrophoresis at 100 V for 45 min. DNA was stained with 0.5 μg/mL ethidium bromide solution (Genesee Scientific, San Diego, USA).

Twenty μl of Protein A Sepharose (Sigma-Aldrich) and 3 μg of the anti-nucleolin antibody were mixed in 1 ml of NT-2 buffer (50 mM Tris HCl; 150 mM NaCl; 1 mM MgCl₂; 0.05% Nonidet P-40) [27 (link)]. The mixture was rotated overnight at 4° C and then washed 3 times with ice-cold NT-2 buffer. HCT116 cells were lysed with lysis buffer (pH 7.5) consisting of 25 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Nonidet P-40, 5% (v/v) glycerol, and 100 U/ml RNase inhibitor (Nacalai Tesque, Tokyo, Japan) [11 (link)]. After these lysates were rotated for 2 h at 4° C with the pre-coated Protein A Sepharose, they were treated with TURBO DNase (Thermo Fisher Scientific) for 10 min at 37° C, and then with 0.1% SDS and proteinase K solution (Wako, Osaka, Japan) for 10 min at 55° C. Finally, bound RNAs were extracted using a phenol-chloroform mixture and then subjected to qPCR.