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Sample GSM3510078 Query DataSets for GSM3510078
Status Public on Dec 31, 2021
Title HBV3-HBcAg
Sample type SRA
Source name HepG2-hNTCP
Organism Homo sapiens
Characteristics hbv genotype: D
agent: Hepatitis B virus
cell line: HepG2-hNTCP
antibody: ChIP anti-HBcAg (DAKO B0586)
Growth protocol HepG2-hNTCP cells (Tropberger et al., PNAS 2015) were grown to confluency in collagen-coated tissue culture flasks (Corning) using DMEM/F12 with 10% FBS and Pen/Strep (all ThermoFisher). Cells were transferred to Williams E medium with Supplement B (CM4000), 0.1 µM Dexamethason (all ThermoFisher) and 2% DMSO (Sigma-Aldrich) two days before infection. Cells were infected with cell culture-derived HBV (Tropberger et al., PNAS 2015) at a multiplicity of infection (MOI) of 1000:1 in the presence of 4% polyethylenglycol (PEG8000; Sigma-Aldrich). Excess virus was washed away 1 day post infection (dpi) with 0.1% Trypsin/EDTA (ThermoFisher) and medium. Cells were maintained with 3 media exchanges per week until 12 dpi to allow establishment of infection.
Extracted molecule genomic DNA
Extraction protocol Cells were fixed in freshly prepared 1% formaldehyde in PBS (both ThermoFisher Scientific) for 5 min prior to quenching with 125 mM glycine in PBS. Pelleted cells were either used directly for analysis (HBV1-3) or snap-frozen and stored at -80°C until use (HBV4-5). Next, cells were washed with cold lysis buffer [PBS with 0.1% Triton X-100, 0.1% NP-40, 1 mM dithiothreitol (DTT), 50 ng/ml trichostatin A (TSA) and 1x EDTA-free protease inhibitor (Roche, Basel, Switzerland)] and lysed in the same buffer for 10 min on ice. Nuclei were pelleted, resuspended in digestion buffer [H2O with 50 mM Tris-HCl pH 7.5, 4 mM MgCl2, 1 mM CaCl2, 10% glycerol, 50 ng/ml TSA, and 1x EDTA-free protease inhibitor (Roche)] and digested with 600 IU/ml micrococcal nuclease (MNase; ThermoFisher Scientific) for 12 min at 37°C. Digestion was stopped by addition of 10 mM EDTA. Nuclei were pelleted at 6500 x g and supernatants collected. The pellet was resuspended in digestion buffer with 10 mM EDTA and 300 mM NaCl and mildly sonicated using a W 375 sonicator (Qsonica, Newtown, CT) at 50% duty cycle and power setting 3. Nuclei were again pelleted, supernatants combined and mixed with an equal amount of sucrose buffer [H2O with 50 mM Tris-HCl pH 7.5, 50 mM NaCl, 5 mM EDTA, 0.01% NP-40, 50 ng/ml TSA, and 1x EDTA-free protease inhibitor (Roche)]. Samples were concentrated using Amicon Ultra-4 100 kDa centrifugal filter units (Millipore-Sigma; Merck KGaA, Darmstadt, Germany) and spun on a 5-30% continuous sucrose gradient in sucrose buffer for 4 h at 40,000 x g and 4°C using a SW41Ti rotor (Beckman Coulter Inc., Brea, CA). Chemicals were obtained from Millipore-Sigma unless noted otherwise. Mononucleosome containing fractions were pooled and concentrated to ~500 µl prior to addition of 100 ng/µl bovine serum albumin (ThermoFisher Scientific). Mononucleosomes were either stored at -20°C or used immediately for ChIP. Antibodies were bound to 20 µl/ChIP Dynabeads Protein G (ThermoFisher) in ChIP buffer [H2O with 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and 200 µg/ml BSA] at 4°C and added to 2 µg of chromatin in ChIP buffer and allowed to bind overnight. Samples were washed six times in LiCl wash buffer [H2O with 150 mM LiCl, 50 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% NP-40, and 0.7% sodium deoxycholate] and eluted in 100 µl elution buffer [H2O with 1% SDS and 100 mM NaHCO3]. Crosslinks were reversed by digestion with Proteinase K (ThermoFisher) for 5 h at 65°C and then digested for another 30 min with DNase-free RNase A (ThermoFisher) at 37°C. DNA was purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
1 ng of DNA was used for sequencing library construction with the KAPA Hyper Prep Kit (Roche) according to the manufacturer’s instructions. For barcoding, 2 µl of TruSeq RNA adapters stocks were used (Illumina, Inc., San Diego, CA). Up to 6 libraries were pooled per sequencing run and 500 ng pooled library subject to HBV-specific target enrichment using the xGen Lockdown Reagents Hybridization and Wash Kit (Integrated DNA technologies, Skokie, IL) according to the manufacturer’s instructions. A custom set of xGen lockdown probes of 60 bp length tiling the entire HBV genome of genotypes A-D was used for target enrichment (Integrated DNA technologies) together with Dynabeads MyOne T-270 Streptavidin (ThermoFisher). The pull-down was amplified for 8 PCR cycles (KAPA HiFi HotStart ReadyMix, Roche) and the product cleaned up using Agencourt AMPure XP beads (Beckman Coulter, Inc.). The product was used to dilute the original library to 20 nM. Sequencing was performed using an Illumina MiSeq sequencer with a MiSeq v3 reagent kit for 2x76 bp paired-end reads (Illumina, Inc.).
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina MiSeq
Description ChIP anti-HBcAg (DAKO B0586)
Data processing base calling: RTA 1.18.54 (from Illumina MCS 2.6.2)
alignment: bowtie2 2.3.0 was used for alignment to 1.1mer viral genomes (with -t -no-unal -no-discordant -no-mixed -I 120 -X 200 option) and all sequences (combined hg38/HBV/phiX; only -t option)
paired read merging: custom script was used to merge paired reads in the output SAM file based on viral genome coordinates. Unsequenced insert was represented as N with low quality score.
BAM conversion: SAM file was converted to BAM, sorted, and indexed using samtools 0.1.19
BEDGRAPH conversion: BAM file was converted to BEDGRAPH using bedtools 2.18.1 (-bg -trackline options)
1mer conversion: custom script was used to wrap around reads that aligned in the overhang of the 1.1mer to the start of the sequence
scaling and wig conversion: custom script was used to convert BEDGRAPH to WIG files while simultaneously scaling it per 106 total reads in the library (hg38+HBV+phiX)
Genome_build: GenBank V01460.1 was used as HBV reference genome (genotype D). GRCh38.p10 was used for the human genome, NC_001422.1 for phiX
Supplementary_files_format_and_content: WIG files contain the number of HBV reads per million total reads for each position of the respective HBV reference genome. Sequences are shifted by -500 nt compared to EcoRI site.
Submission date Dec 12, 2018
Last update date Dec 31, 2021
Contact name Tobias Flecken
Organization name Novartis Institutes for Biomedical Research
Department Infectious Diseases
Street address 5300 Chiron Way
City Emeryville
State/province CA
ZIP/Postal code 94608
Country USA
Platform ID GPL15520
Series (1)
GSE123715 Binding of Nucleolin to HBV cccDNA
BioSample SAMN10585704
SRA SRX5126728

Supplementary file Size Download File type/resource
GSM3510078_HBV3-HBcAg_ayw_wrapped_scaled.wig.gz 612 b (ftp)(http) WIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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