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| Status |
Public on Jun 19, 2024 |
| Title |
HepG2, cccDNA-transfected, DMSO, H3, rep1 |
| Sample type |
SRA |
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| Source name |
HepG2
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| Organism |
Homo sapiens |
| Characteristics |
tissue: HepG2
cell line: Human hepatoma cells
cell type: HBV cccDNA transfection 24 hours before harvest
genotype: H3 (Abcam, #ab1791)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Frozen cell pellets were resuspended in SDS buffer (100mM NaCl, 50mM Tris-HCl pH 8.0, 5mM EDTA, 0.5 % SDS, 1x protease inhibitor cocktail from Roche). The resulting nuclei were spun down, resuspended in the immunoprecipitation buffer at 1mL per 0.5 million cells (SDS buffer and Triton Dilution buffer [100mM NaCl, 100mM Tris-HCl pH 8.0, 5mM EDTA, 5% Triton X-100] mixed in 2:1 ratio with the addition of 1x protease inhibitor cocktail) and processed on a Covaris E220 Focused-ultrasonicator to achieve an average fragment length of 200-300bps with the following parameters: PIP=140, Duty Factor=5, CBP/Burst per sec=200, Time = 1200s. Chromatin concentrations were estimated using the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific) according to the manufacturer’s instructions. The immunoprecipitation reactions were set up in 500uL of the immunoprecipitation buffer in Protein LoBind tubes (Eppendorf) and pre-cleared with 50 µL of Protein G Dynabeads (ThermoFisher Scientific) for two hours at 4 °C. After pre-clearing, the samples were transferred into new Protein LoBind tubes and incubated overnight at 4 °C with the indicated antibodies (Key Resources Table). The next day, 50 µL of BSA-blocked Protein G Dynabeads were added to the reactions and incubated for 2 hours at 4 °C. The beads were then washed two times with low-salt washing buffer (150 mM NaCl, 1 % Triton X-100, 0.1 % SDS, 2 mM EDTA, 20 mM Tris-HCl pH 8.0), two times with high-salt washing buffer (500 mM NaCl, 1 % Triton X-100, 0.1 % SDS, 2 mM EDTA, 20 mM Tris-HCl pH 8.0), two times with LiCL wash buffer (250 mM LiCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 % Na-Deoxycholate, 1 % IGEPAL CA-630) and one time with TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). The samples were then reverse-crosslinked overnight in elution buffer (1 % SDS, 0.1M NaHCO3) and purified using the ChIP DNA Clean & Concentrator kit (Zymo Research) following manufacturer instructions.
After quantification of the recovered DNA fragments, libraries were prepared using the ThruPLEX®DNA-Seq kit (Takara) following the manufacturer’s instructions, purified with SPRIselect magnetic beads (Beckman Coulter), and quantified using a Qubit Flex fluorometer (ThermoFisher Scientific) and profiled with a TapeStation (Agilent).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
adapter trimming (TrimGalore v0.4.5, cutadapt v1.15, FastQC v0.11.5)
alignment to hg38 and HBV genome (bowtie2 v2.3.4.1
deduplication (Picard Tools v2.16.0)
read density profiling (BEDTools v2.29.2)
Assembly: hg38 (human alignment), NC_003977.2 (offset by 500 bp, HBV alignment)
Supplementary files format and content: bigwig
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| Submission date |
Jun 18, 2024 |
| Last update date |
Jun 21, 2024 |
| Contact name |
Andres Mansisidor |
| E-mail(s) |
mansisidor@nyu.edu, mansisidor@rockefeller.edu, amansisi@stevens.edu
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| Organization name |
Rockefeller University
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| Department |
Laboratory of Genome Architecture and Dynamics
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| Lab |
Risca
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| Street address |
1230 York Ave Box 176
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE225716 |
A nucleosome switch primes Hepatitis B Virus infection |
| GSE270130 |
A nucleosome switch primes Hepatitis B Virus infection [ChIP-Seq] |
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| Relations |
| SRA |
SRX24965145 |
| BioSample |
SAMN41895321 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8335293_ChIP_HepG2_DMSO_H3_rep1.hg38.sorted.RmDup.10mNorm.bw |
623.8 Mb |
(ftp)(http) |
BW |
| GSM8335293_ChIP_HepG2_DMSO_H3_rep1_IGO_14046BH_concat_R1_001_val.HBV.PE.sorted.RmDup.10mNorm.ext0.bw |
2.9 Kb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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