Positive charged nylon membrane

DNA samples were added to the denaturation buffer (0.4 mM NaOH and 10 mM EDTA) and denatured at 100°C for 10 min. Furthermore, the samples were chilled on ice for 5 min and applied on a positive-charged nylon membrane (Roche, Basel, Switzerland). The membrane was UV cross-linked and dried for 1 h at 70°C. The membranes were probed with rabbit polyclonal anti-5hmC (1:5,000; cat. no. 39769; Active Motif, Carlsbad, CA, USA) at 4°C overnight. The membranes were then incubated with HRP-conjugated goat anti-rabbit IgG (1:5,000; cat. no. sc-2004; Santa Cruz Biotechnology, Inc.) for 1 h at RT. The target bands were visualized using WesternBright enhanced chemiluminescence reagent (Advansta, Inc., Menlo Park, CA, USA) and the results of the immunoreactions were analyzed with a BioSpectrum^® 500 Imaging System (Ultra-Violet Products Ltd., Cambridge, UK). The membranes were stained with methylene blue (Sigma-Aldrich; Merck KGaA) as a loading control.

HBsAg and HBcAg were determined by CLEIA using commercial assay kits (Fujirebio Inc., Tokyo, Japan). To detect the intracellular replicative intermediates of HBV, the core particle-associated HBV DNA in the cells was isolated and measured by Southern blot[20 (link),21 (link)]. Briefly, cells were harvested and lysed in 1.5 mL of lysis buffer containing 50 mmol/L Tris-HCl (pH 7.4), 1 mmol/L EDTA and 1% IGEPAL CA-630 (Sigma-Aldrich, Japan G.K.). Total cell lysate was treated with 120 μg/mL of RNase A and 30 μg/mL of DNase I for 3 h at 37 °C, in the presence of 6 mmol/L magnesium acetate. HBV DNA was then released by proteinase K digestion, extracted with phenol, and ethanol precipitated. DNA was separated on a 1.2% w/v agarose gel and then transferred to a positive-charged nylon membrane (Roche Diagnostics). The membrane was hybridized with digoxigenin (DIG)-dUTP-labeled full-length HBV genotype C fragment, which was generated using the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche Diagnostics GmbH), and then detected by alkaline phosphatase-labelled anti-DIG antibody according to the manufacturer’s instructions. Signals were analyzed using an ImageQuant LAS 4000 mini (GE Healthcare United Kingdom Ltd).

A 241-bp digoxigenin (DIG)-labeled probe was generated from 100 ng control genomic DNA (gDNA) by PCR reaction using Q5 High-Fidelity DNA Polymerase (NEB) with primers shown in Table S2. Genomic DNA was harvested from control and patient iPSCs using cell lysis buffer (100 mM Tris-HCl pH 8.0, 50 mM EDTA, 1% w/v sodium dodecyl sulfate (SDS)) at 55°C overnight and performing phenol:chloroform extraction. A total of 25 μg of gDNA was digested with AflII at 37°C overnight, run on a 0.8% agarose gel, then transferred to a positive charged nylon membrane (Roche) using suction by vacuum and UV-crosslinked at 120 mJ. The membrane was pre-hybridized in 25 mL DIG EasyHyb solution (Roche) for 3 h at 47°C then hybridized at 47°C overnight in a shaking incubator, followed by two 5-min washes each in 2X Standard Sodium Citrate (SSC) and in 0.1% SDS at room temperature, and two 15-min washes in 0.1x SSC and in 0.1% SDS at 68°C. Detection of the hybridized probe DNA was carried out as described in DIG System User’s Guide. CDP-Star Chemilumnescent Substrate (Sigma-Aldrich) was used for detection and the signal was developed on X-ray film (Genesee Scientific) after 20 to 40 min.