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| Status |
Public on Apr 01, 2022 |
| Title |
4-2 |
| Sample type |
SRA |
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| Source name |
Huh7
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| Organisms |
Homo sapiens; Hepatitis B virus |
| Characteristics |
cell line: Huh7
treatment: transfected HBVcircle-Δcore
chip antibody: anti-H3K4me3 (Cell Signaling Technology, #9751)
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| Treatment protocol |
Huh7 cells were transfected with either HBVcircle-WT or HBVcircle-Δcore, and the cells were harvested at day 3 for HBV cccDNA chromatin immunoprecipitation followed by massive parallel sequencing.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The cccDNA ChIP-Seq assay was performed according to previously described procedures, with slightly modifications. Huh7 cells transfected HBVcircle were cross-linked by 1% formaldehyde (Invitrogen, USA) for 10 min at room temperature, and fixation was quenched by adding 125 mM glycine final concentration for 5 min. Next, the cells were permeated in nuclei isolation buffer [1× PBS with 0.1% Triton X-100, 0.1% NP-40, 1 mM DTT, 10 mM sodium butyrate, 1× protease inhibitor (Roche)] for 10 min at 4℃. Nuclei were pelleted after centrifugation at 9000g for 3 min at 4℃, resuspended and digested in Mnase digestion buffer [50 mM Tris pH 7.5, 4 mM MgCl2, 1 mM CaCl2, 10% (vol/vol) glycerol, 10 mM sodium butyrate, 1× protease inhibitor] with 1500 U/mL Mnase (New England Biolabs, Ipswich, Massachusetts) at 37℃ for 1 h. and digestion was quenched with 10 mM EGTA final concentration on ice. Then, the digested nuclei were harvested by centrifugation at 9000g for 3 min at 4℃, and resuspended by ChIP lysis buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA, 150 mM NaCl, 1% NP-40, 0.1% SDS, 10 mM sodium butyrate, protease inhibitor) with occasional mixing for 2 h at 4℃. The sample was centrifugated at 6500g for 5 min, and the supernatant was collected for immunoprecipitation. For ChIP, the above lysis supernatant was added into protein A/G-conjugated agarose beads after protein A/G-conjugated agarose beads was incubated with indicated antibodies in a rotator overnight at 4℃, and followed by collecting beads and washing five times. Immunocomplexes was eluted in elution buffer (1% SDS, 100 mM NaHCO3), and decross-linked at 65 °C for 5–6 h, the DNA fragments were purified by a TIANamp Genomic DNA Kit (TIANGEN Biotech Co., Beijing, China). For sequencing, the DNA fragments were sent to sequencing company (Annoroad Genomics Company, China).
Repair the ends of DNA fragments, add A to the end of the DNA fragment, adaptors were added into the end of the DNA fragments, PCR amplification, DNA fragments purification.
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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 |
Paired-end sequencing reads in fastq format were mapped to the HBVcircle genome with Bowtie2 in no-discordant and no-mixed mode
Sequencing reads from transfected cells were aligned to the HBVcircle genome and generated the alignment output files (SAM format)
The alignment output files (SAM format) were conversed into the files (BAM format) by using the Samtools
The files (BAM format) were conversed into the files (BW format) by using the Deeptools
ChIP-seq density profiles were generated by operating the alignment output files (BAM format) in the IGV genome browser.
Assembly: reference_sequence.txt
Supplementary files format and content: bam files was generated by using Bowtie2, the files (BAM format) were conversed into the files (BW format) by using the deeptools
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| Submission date |
Mar 29, 2022 |
| Last update date |
Apr 02, 2022 |
| Contact name |
Youquan Zhong |
| E-mail(s) |
2019203010049@whu.edu.cn
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| Organization name |
Wuhan University
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| Department |
TaiKang Medical School,
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| Lab |
XIA lab
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| Street address |
NO.115, Donghu road, Wuchang district, Wuhan city, Hubei province, China.
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| City |
Wuhan |
| State/province |
hubei |
| ZIP/Postal code |
430000 |
| Country |
China |
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| Platform ID |
GPL32090 |
| Series (1) |
| GSE199653 |
Hepatitis B virus core protein is not required for cccDNA transcriptional regulation |
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| Relations |
| BioSample |
SAMN27053234 |
| SRA |
SRX14647690 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5979895_4-2.bw |
2.8 Kb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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