Dnase 1

The pancreatic acinar cells were isolated from 22–30 g male BALB/c mice using collagenase P (Roche) digestion, DNase I (Sangon Biotech), and soybean trypsin inhibitor (Sangon Biotech) [22 (link)]. Cells were resuspended in DMEM/F12 medium (Wisent) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% FBS. After an overnight incubation, “contaminated” cells adhered were discarded, and suspended acinar cells were collected and treated with 10 mM alloxan (Sangon Biotech) for 10 min. For lineage tracing, cells were labeled with 5 μg/mL WGA-FITC (Thermo Fisher Scientific, Waltham, MA, USA) for 24 h and then treated with 100 nM rReg3α. At d 3 and 5, cells were fixed in 4% paraformaldehyde and permeabilized in 0.2% Triton X-100 followed by an incubation with anti-insulin or anti-Ngn3 and Cy5-conjugated secondary antibodies (Table 1). Microscopic images were captured using a Zeiss LSM 5 Pascal laser scanning confocal microscope with 630× magnification.

HBV core DNA was extracted as previously described, with minor modifications [20 (link)]. Briefly, cells were lysed in 200 µL lysis buffer containing 0.5% NP-40, then incubated with Mung Bean Nuclease (M0250S, New England Biolabs, Ipswich, MA, USA) and DNase I (EN0521, Sangon Biotech, Shanghai, China) to remove the input plasmid DNA. Viral DNA was ethanol precipitated after protease K (Calbiochem, San Diego, CA, USA) digestion in the presence of 0.5% SDS at 55 °C overnight. Purified DNA was dissolved in 16 µL double-distilled water and separated by 1% agarose gel. Following overnight transfer to nylon membrane, the blot was hybridized with a Digoxigenin-labeled HBV-specific probe.

RNA isolation and qRT-PCR analysis were performed as previously described [54 (link)]. Briefly, total RNA was isolated using the TRIzol Total RNA Isolation kit (Sangon Biotech, Shanghai, China) and treated with DNase I (Sangon Biotech, Shanghai, China). Eight hundred nanograms of total RNA was reverse-transcribed using RevertAid Premium Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) and diluted ten-fold for PCR amplification. The PCR was performed on a LightCycler480 II instrument (Roche, Basel, Switzerland). Each reaction contained 2 μL of cDNA template, 10 μL of SYBR Green qPCR Master Mix (BBI, Toronto, ON, Canada), and 0.2 μmol L⁻¹ gene-specific primers in a final volume of 20 μL. The PCR conditions included an initial incubation at 95 °C for 3 min, followed by 45 cycles of 95 °C for 5 s and 60 °C for 30 s. The specificity of the PCR reactions was determined by melting curve analyses of the products. Relative expression levels were calculated by the 2^−ΔΔCT method. The rice Actin1 gene was used as the internal control. The primer sequences are listed in Table S9.

Fresh tumor samples were obtained soon after sacrificed of the animals. Tumors were minced and digested with a combination of collagenase I (Sangon,China) and DNase I (Sangon, China). PBMCs were derived from patients with Ficoll (GE Healthcare, USA). The cells were stimulated, stained with membrane markers and applied to MACS column for further analysis.

As previous studies reported,^{50 (link)} to isolate the colon lamina propria cells, colon tissue was cut into small pieces and incubated in epithelial cell solution including HBSS Ca/Mg-Free buffer (Solarbio, China) supplemented with 2% fetal bovine serum, 1 mM DTT and 5 mM EDTA at 37°C shaker for 30 min. The remaining colon pieces were cut into 1 mm pieces and further incubated in enzymatic digestion solution including HBSS buffer supplemented with 400 IU/mL Type Ⅳ collagenase (Sangon, China) and 10 mg/mL DNase I (Sangon, China) for 1 h at 37°C shaker. After complete digestion, the cell suspension was passed through a 200-mesh filter, and then cell suspension was centrifuged at 300 g for 5 min. The isolated colon lamina propria cells were collected for further analysis.

Tissue was surgically minced on a laboratory sterile table, and tissue fragments were preserved in MACS tissue storage until processing.
The tissue samples were processed as described below. Briefly, samples were first washed with phosphate-buffered saline (PBS), minced into small pieces (approximately 1 mm³) on ice, and enzymatically digested with 500 U/ml collagenase I (SangonBiotech), 150 U/ml collagenase II (SangonBiotech), 50 U/ml collagenase IV (SangonBiotech), 0.1 mg/ml hyaluronidase (SangonBiotech), 30 U/ml DNaseI (SangonBiotech), and 5% Fetal Bovine Serum Origin South America (Yeasen) for 60 min at 37°C, with agitation. After digestion, samples were sieved through a 70 μm cell strainer, and centrifuged at 300 g for 5 min. After washing with PBS containing 0.04% BSA, the cell pellets were re-suspended in PBS containing 0.04% BSA and re-filtered through a 35 μm cell strainer. Dissociated single cells were then stained for viability assessment using Calcein-AM (Thermo Fisher Scientific) and Draq7 (BD Biosciences). The single-cell suspension was further enriched with a MACS dead cell removal kit (Miltenyi Biotec).

The colons were longitudinally opened and cut into 1-cm long pieces. The specimens were washed twice in PBS containing 100 U/mL penicillin and 100 μg/mL streptomycin, and then stirred at 37 °C in 1× Hank’s solution containing 5 mM EDTA and 1 mM dithiothreitol for 30 min to remove colonic epithelial cells. The remaining tissues were cut into pieces and digested with HBSS containing 10% FBS (Gbico, #12657-029), 0.5 mg/mL collagenase IV (Sigma, #C5138), and 5 U/mL DNaseI (Sangon Biotech, #A610099, Shanghai, China), at 37 °C for 30 min. The supernatants were then separated by a 40%–70% Percoll density gradient (GE Healthcare, #17-0891-01), and the cells that layered between the 40% and 70% fractions were collected as lamina propria cells.