This [DATASETNAME]readme.txt file was generated on 2024-03-19 by [Alexander Ploss and Yongzhen Liu]



GENERAL INFORMATION

1. Title of Dataset: Development of a dual channel detection system for pan-genotypic simultaneous quantification of hepatitis B and delta viruses

2. Author Information
	A. Principal Investigator Contact Information
		Name: Alexander Ploss
		Institution: Princeton University 
		Address: LTL 110, Washington Road, Department of Molecular Biology, Princeton University, NJ 08544
		Email: aploss@princeton.edu

	B. Associate or Co-investigator Contact Information
		Name: Yongzhen Liu
		Institution: Princeton University 
		Address: LTL 111, Washington Road, Department of Molecular Biology, Princeton University, NJ 08544
		Email: yongzhen@princeton.edu

	C. Alternate Contact Information
		Name: 
		Institution: 
		Address: 
		Email: 

3. Date of data collection (single date, range, approximate date) <suggested format YYYY-MM-DD>: 
2022-07-11 to 2024-03-16

4. Geographic location of data collection <latitude, longitude, or city/region, State, Country, as appropriate>: Princeton University, NJ, USA

5. Information about funding sources that supported the collection of the data: Work in the Ploss lab is supported by grants from the National Institutes of Health (R01 AI138797, R01 AI107301, R01 AI146917, R01 AI153236), funding from OpenPhilantrophy and Princeton University (all to A.P.). The Molecular Biology Flow Cytometry Resource Facility is partially supported by the Cancer Institute of New Jersey Cancer Center Support grant (P30CA072720). This work utilized NIH S10 Shared Instrumentation Grants S10-OD026983 and S10-OD030269 (awarded to N.A.C). Y.L. was supported by a postdoctoral fellowship from the New Jersey Commission on Cancer Research (NJCCR, OCR24PDF002). S.M. was supported by the National Institute of General Medicine Sciences of the National Institutes of Health under Award Number T32GM007388 and a predoctoral NJCCR fellowship. 


SHARING/ACCESS INFORMATION

1. Licenses/restrictions placed on the data: CC-BY 4.0

2. Links to publications that cite or use the data: not yet available 

3. Links to other publicly accessible locations of the data: https://doi.org/10.34770/s4zx-ab39

4. Links/relationships to ancillary data sets: N/A

5. Was data derived from another source? no
	



DATA & FILE OVERVIEW

1. File List: 
<list all files (or folders, as appropriate for dataset organization) contained in the dataset, with a brief description>

Raw data-Development of a dual channel detection system for pan-genotypic simultaneous quantification of hepatitis B and delta viruses.xlsx

2. Relationship between files, if important: N/A

3. Additional related data collected that was not included in the current data package: No

4. Are there multiple versions of the dataset? No
	A. If yes, name of file(s) that was updated: 
		i. Why was the file updated? 
		ii. When was the file updated? 


METHODOLOGICAL INFORMATION

1. Description of methods used for collection/generation of data: 
<Include links or references to publications or other documentation containing experimental design or protocols used in data collection>

HBV and HDV qPCR primers were designed in genomic regions that are highly conserved among different genotypes obtained from Azenta Life Sciences (Burlington, MA). Both HBV and HDV probes were designed based on Affinity PlusTM qPCR probe technology to impart heightened structural stability, leading to increased hybridization melt temperature (Tm) and were obtained from Integrated DNA Technologies (Coralville, IA). For the One-Step quantitative PCR, the Luna® Universal Probe One-Step RT-qPCR kit was used at a final concentration of 0.4 uM primers and 0.2 uM probes in the single reaction system for HBV and HDV, respectively. The PCR was performed at 55˚C for 10 mins, 95 ˚C for 1 min followed by 45-50 cycles at 95 ˚C for 15s, and 61 ˚C for 40s for signal capture. 

The HBV DNA and HDV RNA sequences of different genotypes were retrieved from the National Center for Biotechnology Information (NCBI) and three representative genomes were used for each genotype except for HDV genotypes 1 and 6 for which an insufficient number of genome sequences were available. The phylogenic trees were built by MegAlign Pro (DNASTAR Inc., Madison, WI) using a RAXML alignment of HBV DNA or HDV RNA sequences among genotypes, respectively. 

Detection and quantification of HBsAg and HBeAg levels were performed by a chemiluminescence immunoassay (CLIA) according to the manufacturer’s instructions (Autobio Diagnostics CO., LTD, Zhengzhou, Henan, China) and the chemiluminescence was determined by a TriStar Multimode Reader (Berthold Technologies, Bad Wildbad, Germany). 

The HBVcircle used in this study was prepared by using the parental construct in combination with the minicircle E. coli strain which has been specifically engineered to express the phi 31 integrase and Scel endonuclease upon arabinose induction. This method was used by our lab previously (Wei, L. & Ploss, A. Core components of DNA lagging strand synthesis machinery are essential for hepatitis B virus cccDNA formation. Nat Microbiol 5, 715-726, doi:10.1038/s41564-020-0678-0 (2020)). The HBV genotype plasmids were prepared by our lab previously (Yongzhen Liu, Debby Park, Thomas R. Cafiero, Yaron Bram, Vasuretha Chandar, Anna Tseng, Hans P. Gertje, Nicholas A. Crossland, Lishan Su, Robert E. Schwartz, Alexander Ploss. JHEP Rep
. 2022 Jul 9;4(9):100535. doi: 10.1016/j.jhepr.2022.100535.)

Serum samples from deidentified HBV+ patients were obtained from the American Red Cross, and samples from HBV+/HDV+ patients from ID Care. This work was deemed non-human subjects research by the Princeton University Institutional Review Board (IRB).

Human albumin ELISA for assessment of human hepatocyte engraftment of chimeric mice. Levels of human albumin in mouse serum were quantified by ELISA as described previously20. Briefly, 96-well flat-bottomed plates (Nunc, Thermo Fisher Scientific, Waltham, MA) were coated with goat anti-human albumin antibody (1:500, #A80-129A, Thermo Fisher Scientific, Waltham, MA) in coating buffer (1.59 g Na2CO3, 2.93 g NaHCO3, 1L dH2O, pH = 9.6) for 1 h at 37 ˚C. The plates were washed four times with wash buffer (0.05% (vol/vol) Tween 20 (Sigma Aldrich, St. Louis, MO) in 1X PBS), and then incubated with superblock buffer (Fisher Scientific, Hampton, NH) for 1 h at 37 ˚C. Plates were washed twice. Human serum albumin (Sigma Aldrich, St. Louis, MO) was diluted to 1 μg/mL in sample diluent (10% Superblock, 90% wash buffer), then serially diluted 1:2 in 135 μL sample diluent to establish an albumin standard. Mouse serum (5 μl) was used for a 1:10 serial dilution in 135 μL sample diluent. The coated plates were incubated for 1 h at 37 ˚C, then washed three times. Mouse anti-human albumin (50 μL, 1:2000 in sample diluent, Abcam, Cambridge, UK) was added and plates were incubated for 2 h at 37 ˚C. Plates were washed four times and 50 μL of goat anti-mouse-horse radish peroxidase (HRP, 1:10,000 in sample diluent, LifeTechnologies, Carlsbad, CA) were added and incubated for 1 h at 37 ˚C. Plates were washed six times. Tetramethylbenzidine (TMB, 100 μL) substrate (Sigma Aldrich, St. Louis, MO) was added and the reaction was stopped with 25 μL 2-N H2SO4. Absorbance was read at 450λ on the BertholdTech TriStar (Bad Wildbad, Germany).

Phylogenetic analysis of HBV and HDV genome variety among genotypes. The HBV DNA and HDV RNA sequences of different genotypes were retrieved from the National Center for Biotechnology Information (NCBI) and three representative genomes were used for each genotype except for HDV genotypes 1 and 6 for which an insufficient number of genome sequences were available. The phylogenic trees were built by MegAlign Pro (DNASTAR Inc., Madison, WI) using a RAXML alignment of HBV DNA or HDV RNA sequences among genotypes, respectively. The nucleotide numbering is based on the genome in the first line and different nucleotides are colored.

Quantification of HBV pgRNA by quantitative RT-PCR. HBV RNA purified from supernatants was used to determine extracellular nucleocapsid-associated HBV pre-genome RNA (pgRNA) level as described previously21. Briefly, 7.5 ul of isolated nucleic acid was treated with DNase I (Thermo Fisher Scientific, Waltham, MA) followed by reverse transcription with a specific HBV primer (5’-CGAGATTGAGATCTTCTGCGAC-3’, nt 2415-2436, numbered based on gt D with GenBank accession no. U95551.1) located in precore/core region21 and anchored sequences using RevertAidTM First Strand DNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). For quantitative assays, the standards with 1-mer HBV target template were cloned into the TOPO-Blunt Cloning vector (Thermo Fisher, Waltham, MA, USA #450245) and the copy number was calculated based on the number of nucleotides of the plasmid and concentration of the plasmid preparation. A master mix was created containing 15 µl 2x Taqman reaction mix (Applied Biosystems, Waltham, MA, USA), 500 nM forward and reverse primers, 200 nM probe and 3 µl synthesized cDNA in a 30 µL reaction. The master mix was then added to the samples or to the 10-fold serial dilution standards and the following cycling program was used to run the qPCR: 95 ºC for 10 min; 45 cycles at 95 ºC for 15 sec and 58 ºC for 45 sec. 

Western blotting. Cells which had been seeded in a 24-well plate were lysed with 150 μL/well of 1X NuPAGE LDS buffer (Life Technologies, Carlsbad, CA) containing 2.5% (vol/vol) 2-mercaptoethanol (β-ME) at room temperature for 10 mins. The cell lysate was then transferred to a sterile 1.5 ml tube and heat-inactivated at 95 ˚C for 20 min. The samples were then loaded and separated by 10% (w/vol) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a nitrocellulose membrane (Roche, Basel, Switzerland). The membrane was blocked with 5% (w/vol) skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature and then incubated overnight at 4 ˚C with the HBV core protein primary antibody (1:1000 dilution, rabbit polyclonal antibody, kindly provided by Dr. Ju-Tao Guo, Blumberg Institute, Doylestown, PA) or HDAg antibody (1:1000 dilution, rabbit polyclonal antibody, a gift from John Taylor, Fox Chase Cancer Center, Philadelphia, PA). Next, the membranes were washed three times with 1X (Tris-Buffered Saline, 0.1% Tween® 20 Detergent) TBST and incubated with a goat anti-Rabbit IgG (1:5,000 dilution; Thermo Fisher Scientific, Waltham, MA) secondary antibody at room temperature for 1 hour. After further washing with TBST, the signal intensities of the membranes were scanned and visualized using an Odyssey CLx imager (Li-COR Biosciences, Lincoln, NE). The signal quantification was performed by ImageJ.

For immunofluorescence of HBcAg and HDAg of the infection samples, HepG2 cells +/- NTCP were grown on collagen precoated cover slides and fixed 6 days after infection with 4% (vol/vol) paraformaldehyde (PFA). For transfection samples, HepG2 cells were grown on collagen precoated cover slides and fixed 3 days after transfection with 4% (vol/vol) PFA. The cells were then washed 3 times with 1xPBS followed by 1 h of incubation with blocking and permeabilization buffer (5% BSA, 5% FBS, 0.3% Triton-X-100 in 1PBS, all (vol/vol)) at room temperature. Thereafter, cells were incubated with either anti-HBc antibody (1:500 dilution, #B0586, Dako, Denmark) or anti-HDAg (1:250 dilution, a gift from John Taylor, Fox Chase Cancer Center, Philadelphia, PA), respectively in the dilution buffer (1% BSA, 1% FBS, 0.3% Triton-X-100 all (vol/vol) in 1xPBS) at 4 C overnight, washed for 3 times and incubated with an Alexa Fluor 488 conjugated goat anti-rabbit secondary antibody (1:1000 dilution, #A11008, Thermo Fisher Scientific, Waltham, MA) and DAPI (1 µg/mL) in dilution buffer (1% BSA, 1% FBS, 0.3% Triton-X-100 all (vol/vol) in 1xPBS) at room temperature for 1 h. After 3 washes, the cover slides with cells were then transferred onto a glass slide with 2 µl ProLong Gold antifade solution (#P36930, Thermo Fisher Scientific, Waltham, MA) and sealed with CoverGrip™ Coverslip Sealant (#23005, Biotium Inc., Fremont, CA). The slides were then covered with foil and dried overnight at 4 ºC and subsequently analyzed with a confocal microscope (Nikon A1R-STED, Nikon, Melville, NY) in the Imaging Core facility at Princeton University. 

Tissue samples were fixed for a minimum of 72 h in 4% (w/vol) paraformaldehyde (PFA) before processing in a Tissue-Tek VIP-5 automated vacuum infiltration processor (Sakura Finetek USA, Torrance, CA) and embedded in paraffin using a HistoCore Arcadia paraffin embedding machine (Leica, Wetzlar, Germany). 5-μm tissue sections were generated using a RM2255 rotary microtome (Leica) and transferred to positively charged slides, deparaffinized in xylene, and dehydrated in graded ethanol. A Ventana Discovery Ultra tissue autostainer (Roche, Basel, Switzerland) was used for multiplex fluorescent immunohistochemistry (mfIHC). Briefly, tyramide signal amplification (TSA) was used in an iterative approach to covalently bind Opal fluorophores (Akoya Bioscience, Marlborough, MA) to tyrosine residues in tissue. Sequential heat-stripping was performed on each primary-secondary antibody complex following each fluorophore application until all antibodies were developed. Liver tissue from non-humanized and non-infected FNRG mice were used as a negative control for the immunohistochemistry assay to confirm the specificity of antibodies. 

Fluorescently labeled slides were imaged using a Vectra PolarisTM Quantitative Pathology Imaging System (Akoya Biosciences, Marlborough, MA). Exposures for all Opal dyes were established utilizing regions of interest with strong signal intensities to minimize exposure times and maximize the specificity of the signal detected. To maximize signal-to-noise ratios, images were spectrally unmixed using a synthetic library specific for each Opal fluorophore and DAPI. Furthermore, an unstained section was used to create an autofluorescence signature that was subsequently removed from whole-slide images using InForm software version 2.4.8 (Akoya Biosciences, Marlborough, MA). InForm exported the multispectrally unmixed images as QPtiffs, which were then fused together as a single whole slide image in HALO (Indica Labs, Albuquerque, NM) for acquisition of representative images.

The correlation and regression analysis of the quantifications between HBV-HDV dual and the HBV or HDV single detection system were performed using GraphPad Prism (Version 10.1.1, Boston, MA) software. Simple linear regression was used and goodness of fit was characterized by the R squared values from the Pearson r. 95% confidence intervals shown as dashed lines on the graphs. 


2. Methods for processing the data: 
<describe how the submitted data were generated from the raw or collected data>

Raw data in the file are the direct output from the assays and have not be further processed. 

3. Instrument- or software-specific information needed to interpret the data: 
<include full name and version of software, and any necessary packages or libraries needed to run scripts>
Softwares that can open CSV or Jpg files are needed to open the data sheet.

4. Standards and calibration information, if appropriate: 
For the ELISA assays for HBsAg, six standards from A to F were included in the ELISA assays and their concentration is 0, 0.05, 0.8, 10, 85 and 250 IU/mL. For the ELISA assays for HBeAg, six standards from A to F were included in the ELISA assays and their concentration is 0, 0.1, 0.7, 5, 35 and 200 PEI U/mL. For the quantification assays for HBV DNA, HDV RNA or HBV pgRNA, the standards from 10, 100, 1000, 10,000, 1000,00, 1000,000, 10,000,000 copies/ul were used.

5. Environmental/experimental conditions: 
The conditions are described in the methods part in this file and in the published paper. 

6. Describe any quality-assurance procedures performed on the data: 
For the ELISA and qPCR assays, a standard curve will be made and only if the R square of the standard curve is larger than 0.9 then the standard curve is treated as reliable. 

7. People involved with sample collection, processing, analysis and/or submission: 
Yongzhen Liu, Stephanie Maya, Sebastian Carver, Aoife K. O’Connell, Anna Zen, Hans P. Gertje, Kathleen Seneca, Ronald G. Nahass, Nicholas A. Crossland, Alexander Ploss

DATA-SPECIFIC INFORMATION FOR: [Figure 1 sheet-Fig.1B&C]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 2

2. Number of cases/rows: 7

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>
HBV input, copies/uL; Detected HBV concentration, log10 copies/uL; Detected HDV concentration, log10 copies/uL;
4. Missing data codes: undetected
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Figure 1 sheet-Fig.1D]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 2

2. Number of cases/rows: 7

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>
HBV input, copies/uL; HDV input, copies/uL; Detected HBV concentration, log10 copies/uL; Detected HDV concentration, log10 copies/uL;
4. Missing data codes: undetected

<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Figure 1 sheet-Fig.1F]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 2

2. Number of cases/rows: 7

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>
HBV input, copies/uL; HDV input, copies/uL; Detected Ct values;

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not available)


DATA-SPECIFIC INFORMATION FOR: [Figure 1 sheet-Fig.1G]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 2

2. Number of cases/rows: 25

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>
Detected HBV concentrations by HBV single detection system, log10 copies/uL; 
Detected HBV concentrations by HBV-HDV dual detection system, log10 copies/uL; 

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Figure 1 sheet-Fig.1H]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 2

2. Number of cases/rows: 21

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>
Detected HDV concentrations by HDV single detection system, log10 copies/uL; 
Detected HBV concentrations by HBV-HDV dual detection system, log10 copies/uL; 

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Figure 1 sheet-Fig.1I]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 5

2. Number of cases/rows: 4

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>
Mock transfection;
HBV monotransfection;
HBV-HDV cotransfection
Detected HDV concentrations by HBV-HDV dual detection system, log10 copies/uL; 
Detected HBV concentrations by HBV-HDV dual detection system, log10 copies/uL; 

4. Missing data codes: undetected
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Figure 2 sheet-Fig.2C]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 2

2. Number of cases/rows: 5

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

Without DNase I treatment, detected HBV concentrations by HBV-HDV dual detection system, log10 copies/uL; 
With DNase I treatment, Detected HBV concentrations by HBV-HDV dual detection system, log10 copies/uL; 

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Figure 2 sheet-Fig.2D]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 2

2. Number of cases/rows: 6

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

Without DNase I treatment, detected HBV concentrations by HBV-HDV dual detection system, log10 copies/uL; 
With DNase I treatment, Detected HBV concentrations by HBV-HDV dual detection system, log10 copies/uL; 

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Figure 2 sheet-Fig.2E]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 9

2. Number of cases/rows: >=3

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

Mock transfection, detected HBV concentrations by HBV-HDV dual detection system, log10 copies/uL; 
Genotype A plasmid transfection, detected HBV concentrations by HBV-HDV dual detection system, log10 copies/uL; 
Genotype B plasmid transfection, detected HBV concentrations by HBV-HDV dual detection system, log10 copies/uL; 
Genotype C plasmid transfection, detected HBV concentrations by HBV-HDV dual detection system, log10 copies/uL; 
Genotype D plasmid transfection, detected HBV concentrations by HBV-HDV dual detection system, log10 copies/uL;
Genotype E plasmid transfection, detected HBV concentrations by HBV-HDV dual detection system, log10 copies/uL;  
Genotype F plasmid transfection, detected HBV concentrations by HBV-HDV dual detection system, log10 copies/uL; 
Genotype G plasmid transfection, detected HBV concentrations by HBV-HDV dual detection system, log10 copies/uL; 
Genotype H plasmid transfection, detected HBV concentrations by HBV-HDV dual detection system, log10 copies/uL; 

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Figure 3 sheet-Fig.3A]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 10

2. Number of cases/rows: >=5

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>


HBV genotype A plasmid transfection, HBV DNA levels, copies/mL; 
HBV genotype A plasmid + HDV transfection, HBV DNA levels, copies/mL; 

HBV genotype B plasmid transfection, HBV DNA levels, copies/mL; 
HBV genotype B plasmid + HDV transfection, HBV DNA levels, copies/mL; 

HBV genotype C plasmid transfection, HBV DNA levels, copies/mL; 
HBV genotype C plasmid + HDV transfection, HBV DNA levels, copies/mL; 

HBV genotype D plasmid transfection, HBV DNA levels, copies/mL; 
HBV genotype D plasmid + HDV transfection, HBV DNA levels, copies/mL; 

HDV plasmid transfection, HBV DNA levels, copies/mL; 
HBV preS1 plasmid + HDV transfection, HBV DNA levels, copies/mL; 

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Figure 3 sheet-Fig.3B]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 10

2. Number of cases/rows: 8

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>


HBV genotype A plasmid transfection, HDV levels, copies/mL; 
HBV genotype A plasmid + HDV transfection, HDV levels, copies/mL; 

HBV genotype B plasmid transfection, HDV levels, copies/mL; 
HBV genotype B plasmid + HDV transfection, HDV levels, copies/mL; 

HBV genotype C plasmid transfection, HDV levels, copies/mL; 
HBV genotype C plasmid + HDV transfection, HDV levels, copies/mL; 

HBV genotype D plasmid transfection, HDV levels, copies/mL; 
HBV genotype D plasmid + HDV transfection, HDV levels, copies/mL; 

HDV plasmid transfection, HDV levels, copies/mL; 
HBV preS1 plasmid + HDV transfection, HDV levels, copies/mL; 

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Figure 3 sheet-Fig.3C]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 10

2. Number of cases/rows: 4

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>


HBV genotype A plasmid transfection, HBsAg levels, IU/mL; 
HBV genotype A plasmid + HDV transfection, HBsAg levels, IU/mL; 

HBV genotype B plasmid transfection, HBsAg levels, IU/mL; 
HBV genotype B plasmid + HDV transfection, HBsAg levels, IU/mL; 

HBV genotype C plasmid transfection, HBsAg levels, IU/mL; 
HBV genotype C plasmid + HDV transfection, HBsAg levels, IU/mL; 

HBV genotype D plasmid transfection, HBsAg levels, IU/mL; 
HBV genotype D plasmid + HDV transfection, HBsAg levels, IU/mL; 

HDV plasmid transfection, HBsAg levels, IU/mL; 
HBV preS1 plasmid + HDV transfection, HBsAg levels, IU/mL; 

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Figure 3 sheet-Fig.3D]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 10

2. Number of cases/rows: 4

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>


HBV genotype A plasmid transfection, HBeAg levels, PEI U/mL; 
HBV genotype A plasmid + HDV transfection, HBeAg levels, PEI U/mL; 

HBV genotype B plasmid transfection, HBeAg levels, PEI U/mL; 
HBV genotype B plasmid + HDV transfection, HBeAg levels, PEI U/mL; 

HBV genotype C plasmid transfection, HBeAg levels, PEI U/mL; 
HBV genotype C plasmid + HDV transfection, HBeAg levels, PEI U/mL; 

HBV genotype D plasmid transfection, HBeAg levels, PEI U/mL; 
HBV genotype D plasmid + HDV transfection, HBeAg levels, PEI U/mL; 

HDV plasmid transfection, HBeAg levels, PEI U/mL; 
HBV preS1 plasmid + HDV transfection, HBeAg levels, PEI U/mL; 

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Figure 3 sheet-Fig.3E]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 12

2. Number of cases/rows: 2 western repeats, 2 quantification for each blot

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

HBc, HDAg, Actin levels under mock, HDV, HDV+HBV-2.7, HBV-gtA, HBV-gtA+HDV,HBV-gtB, HBV-gtB+HDV,
HBV-gtC, HBV-gtC+HDV, HBV-gtD, HBV-gtD+HDV, HBV-gtE, HBV-gtE+HDV, HBV-gtG, HBV-gtG+HDV

4. Missing data codes: the blots were removed from the final figure 3E because the antibody used doesn't recognize the HBc from genotype E and G.
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Fig 3E western blot images]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 12

2. Number of cases/rows: 2 western repeats, 2 quantification for each blot

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

HBc, HDAg, Actin levels under mock, HDV, HDV+HBV-2.7, HBV-gtA, HBV-gtA+HDV,HBV-gtB, HBV-gtB+HDV,
HBV-gtC, HBV-gtC+HDV, HBV-gtD, HBV-gtD+HDV, HBV-gtE, HBV-gtE+HDV, HBV-gtG, HBV-gtG+HDV

4. Missing data codes: N/A (not applicable).
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Fig 3E western blot images-repeat experiment]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 12

2. Number of cases/rows: 2 western repeats, 2 quantification for each blot

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

HBc, HDAg, Actin levels under mock, HDV, HDV+HBV-2.7, HBV-gtA, HBV-gtA+HDV,HBV-gtB, HBV-gtB+HDV,
HBV-gtC, HBV-gtC+HDV, HBV-gtD, HBV-gtD+HDV, HBV-gtE, HBV-gtE+HDV, HBV-gtG, HBV-gtG+HDV

4. Missing data codes: N/A (not applicable).
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)


DATA-SPECIFIC INFORMATION FOR: [Figure 4 sheet-Fig.4A]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 2

2. Number of cases/rows: 4

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

HepG2, HepG2-NTCP

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A

DATA-SPECIFIC INFORMATION FOR: [Fig 4B and C-orginal images]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 4

2. Number of cases/rows: 5

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

Nuclei, NTCP-RFP, HBcAg/HDAg, Merge

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A

DATA-SPECIFIC INFORMATION FOR: [Figure 4 sheet-Fig.4D]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 7

2. Number of cases/rows: 3

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

HepG2-Mock, HepG2-NTCP-HBV&HDV coinfection, HepG2-NTCP-HDV mono infection, HepG2-NTCP-HBV mono infection, day3, day5, day7

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Figure 4 sheet-Fig.4E]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 6

2. Number of cases/rows: >=2

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

Mock, HDV mono infection, HBV mono infection, HBV&HDV coinfection, day5, day7

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Figure 4 sheet-Fig.4F]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 6

2. Number of cases/rows: 4

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

Mock, HDV mono infection, HBV mono infection, HBV&HDV coinfection, day5, day7

4. Missing data codes: the HDV RNA levels in Mock, HDV mono infection, HBV mono infection groups are undetectable.
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Figure 5 sheet-Fig.5C]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 13

2. Number of cases/rows: 1

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

#2460, #2445, #2446, #2444, #157, #89, #391, Baseline (BL), W2, W3, W5, W7, W9

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)


DATA-SPECIFIC INFORMATION FOR: [Figure 5 sheet-Fig.5D]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 12

2. Number of cases/rows: 1

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

#2460, #2445, #2446, #2444, #157, #89, #391, Baseline (BL), W3, W5, W7, W9

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Figure 5 sheet-Fig.5E]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 12

2. Number of cases/rows: 1

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

#89-HBV&HDV, #391-HBV&HDV, #157-HBV&HDV, #2444-HBV, #2445-HBV, #2446-HBV, #2460-mock, Baseline (BL), W3, W5, W7, W9

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Figure 5 sheet-Fig.5F]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 12

2. Number of cases/rows: 1

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

#89-HBV&HDV, #391-HBV&HDV, #157-HBV&HDV, #2444-HBV, #2445-HBV, #2446-HBV, #2460-mock, Baseline (BL), W3, W5, W7, W9

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Figure 6 sheet-Fig.6A]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 14

2. Number of cases/rows: 1

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

ARC_patient_1	ARC_patient_2	ARC_patient_3	ARC_patient_4	ARC_patient_5	ARC_patient_6	ARC_patient_7	ARC_patient_8	ARC_patient_9	ARC_patient_10	ID_patient_1	ID_patient_2	ID_patient_3	ID_patient_4

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Figure 6 sheet-Fig.6B]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 14

2. Number of cases/rows: 1

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

ARC_patient_1	ARC_patient_2	ARC_patient_3	ARC_patient_4	ARC_patient_5	ARC_patient_6	ARC_patient_7	ARC_patient_8	ARC_patient_9	ARC_patient_10	ID_patient_1	ID_patient_2	ID_patient_3	ID_patient_4

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Figure 6 sheet-Fig.6D]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 14

2. Number of cases/rows: 2

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

ARC_patient_1	ARC_patient_2	ARC_patient_3	ARC_patient_4	ARC_patient_5	ARC_patient_6	ARC_patient_7	ARC_patient_8	ARC_patient_9	ARC_patient_10	ID_patient_1	ID_patient_2	ID_patient_3	ID_patient_4

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Figure 6 sheet-Fig.6E]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 14

2. Number of cases/rows: 2

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

ARC_patient_1	ARC_patient_2	ARC_patient_3	ARC_patient_4	ARC_patient_5	ARC_patient_6	ARC_patient_7	ARC_patient_8	ARC_patient_9	ARC_patient_10	ID_patient_1	ID_patient_2	ID_patient_3	ID_patient_4

4. Missing data codes: undetectable
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Supplementary Figure 1 sheet]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 4

2. Number of cases/rows: >=3

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

HBV DNA, HBsAg, HBVcircle, mock

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Supplementary Figure 2-HBV DNA.csv]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 10

2. Number of cases/rows: 1

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

HBV DNA levels from 7 to 1 Log10 copies/ul, Ct value

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Supplementary Figure 2-HBV DNA.jpg]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 10

2. Number of cases/rows: 1

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

HBV DNA levels from 7 to 1 Log10 copies/ul, HDV RNA levels from 7 to 1 Log10 copies/ul, Ct value

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Supplementary Figure 2-HDV RNA.csv]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 10

2. Number of cases/rows: 1

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

HDV RNA levels from 7 to 1 Log10 copies/ul, Ct value

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Supplementary Figure 2-HDV RNA.jpg]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 10

2. Number of cases/rows: 1

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

HDV RNA levels from 7 to 1 Log10 copies/ul, Ct value

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Supplementary Figure 4 sheet-4A]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 2

2. Number of cases/rows: >=4

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

Mock, HBV-2.7

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)

DATA-SPECIFIC INFORMATION FOR: [Supplementary Figure 4 sheet-4B]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 10

2. Number of cases/rows: 8

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

genotypeA-HBV	genotypeA-HBV-HDV	genotypeB-HBV	genotypeB-HBV-HDV	genotypeC-HBV	genotypeC-HBV-HDV	genotypeD-HBV	genotypeD-HBV-HDV	HDV	HDV-HBV preS1

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: N/A (not applicable)


DATA-SPECIFIC INFORMATION FOR: [Supplementary Figure 4 sheet-4C]
<repeat this section for each dataset, folder or file, as appropriate>

1. Number of variables: 11

2. Number of cases/rows: 4

3. Variable List: 
<list variable name(s), description(s), unit(s)and value labels as appropriate for each>

Mock, HDV,	HDV-HBV preS1, genotypeA-HBV	genotypeA-HBV-HDV	genotypeB-HBV	genotypeB-HBV-HDV	genotypeC-HBV	genotypeC-HBV-HDV	genotypeD-HBV	genotypeD-HBV-HDV	

4. Missing data codes: N/A
<list code/symbol and definition>n

5. Specialized formats or other abbreviations used: normalized western blot intensity