Phenol chloroform

Cells (8 × 10⁴) were plated on a collagen-coated 35 mm dish. Following treatments, cells were permeabilized with CSK100 for 10 min at RT, gently washed with MNase buffer (50 mM Tris-HCl pH 8.0, 5 mM NaCl, 5 mM CaCl₂), and then treated with the 75 gelU/ml of MNase (New England Biolab, Cat# M0247S) in MNase buffer for 5 ~ 20 min at 25 °C. The MNase-treated cells were lysed in 250 μl of TES buffer (10 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.5% SDS). The cell lysates were sequentially treated with 1 mg/ml RNaseA (NipponGene, Cat# 318-06391) for 45 min and with 200 μg/ml Proteinase K (QIAGEN, Cat# 19133) for 90 min at 50 °C, after which an equal volume of phenol/chloroform (NipponGene, Cat# 311-90151) was added. The insoluble fraction was pelleted at 20,000 x g, the supernatant was mixed with 0.5 M of NaCl, and an EtOH wash was performed. The extracted DNA was resuspended in TE buffer (pH 8.0), resolved by 1.2% agarose gel (Fast Gene, Cat# NE-AG02) electrophoresis at 1 μg per lane, and stained with EtBr (NipponGene, Cat# 315-90051). Images were captured with a LAS 3000 luminescent image analyzer (FujiFilm) for intensity analysis with ImageJ.

The fresh porcine small intestine, urinary bladder, and liver were purchased from a local slaughterhouse (Tokyo Shibaura Zouki, Tokyo, Japan) and stored at 4 °C until further use. Normal saline (0.9% w/v) was obtained from Otsuka Pharmaceuticals (Tokyo, Japan). DNase I and RNase were purchased from Roche Diagnostics (Basel, Switzerland). Magnesium chloride hexahydrate was purchased from Merck (Darmstadt, Germany). Phosphate-buffered saline (PBS), sodium chloride (NaCl), sodium dodecyl sulfate (SDS), neutral-buffered (pH 7.4) solution of 10% formalin, protease-K, 0.1 mol/L sodium citrate hydroxide solution, anhydrous ethanol, and Eagle′s minimal essential medium were purchased from FUJIFILM Wako Pure Chemical Corp. (Osaka, Japan). Phenol/chloroform was purchased from Nippon Gene (Tokyo, Japan). Tris (hydroxymethyl) aminomethane (Tris), citric acid, and pepsin (derived from porcine gastric mucosa, ≥2500 units/mg) were obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA) 0.01 N HCl solution was purchased from Nacalai Tesque (Kyoto, Japan). HH cells were purchased from the Japanese Collection of Research Bioresources Cell Bank (JCRB Cell Bank).