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| Status |
Public on Mar 03, 2023 |
| Title |
MNase-seq,HEK293T cells, mock transfection, t0, 0250U |
| Sample type |
SRA |
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| Source name |
HEK293T
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
cell type: Human embryonic kidney cells
genotype: WT
treatment: mock transfection
treatment: 00 hr, 250 Units MNase
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
In vivo MNase-seq was performed on purified nuclei at different timepoints after transfection with HBV cccDNA or on in vitro reconstituted chromatin.
For MNase-seq on in vitro reconstituted chromatin, titration of MNase was performed to determine enzyme concentrations that yielded partial- , fully-, or over-digested chromatin arrays. Chromatinized DNA (100 ng) was digested using MNase (NEB) in 25 µL reactions with 2.5–25 units of enzymes that was incubated for 5 minutes at 37C before 1 mM of CaCl2 was added and incubation continued for another 5 minutes at 37C. The reaction was stopped by adding 20 mM EDTA and 1% SDS. DNA was subsequently purified using Clean and Concentrated-5 DNA columns (Zymo). Sequencing libraries were prepared using NEBNext Ultra II DNA Library Prep Kit – DNA repair, End Prep, USER, and Ligation of Unique Dual Index Barcodes or Unique Dual Index UMI Adaptors (NEB). Barcoding PCR was consistently carried out using only 5 PCR cycles with NEBNext High-Fidelity 2X PCR Mix and subsequent size selection using agarose gel or beads (Ampure XP). For MNase-seq on cell cultured samples, HEK293T cells were transfected with recombinant cccDNA 4 or 24-hours prior to harvest for MNase-seq. The nuclei of HEK293 cells were purified by incubating cells in Nuclei isolation buffer for 10 minutes (0.1% Igepal, 0.1% Triton X -100, 1 mM DTT in 1 X PBS), mechanically lysing with 10 strokes of loose (A) pestle Dounce homogenizer, and washing nuclei twice in 10 mL of Wash Buffer with Tween (10 mM Tris-HCl pH 7.5, 2.5 mM NaCl, 3 mM MgCl2, and 0.1% Tween-20). Titration of MNase was similarly performed to determine enzyme concentrations that yielded partial- , fully-, or over-digested genomic DNA. MNase (NEB) was added to 2 x 106 nuclei in 50 µL reaction volume with 250–2000 units of enzyme that was incubated for 5 minutes at 37 C before 1 mM of CaCl2 was added and incubation continued for another 5 minutes at 37 C. The reaction was stopped by adding 20 mM EDTA and 1 % SDS. DNA was subsequently purified using Clean and Concentrated-5 DNA columns (Zymo). Sequencing libraries were prepared using NEBNext Ultra II DNA Library Prep Kit – DNA repair, End Prep, USER, and Ligation of Unique Dual Index UMI Adaptors (NEB). Barcoding PCR was consistently carried out with NEBNext High-Fidelity 2X PCR Mix and subsequent size selection using agarose gel or beads (Ampure XP). Libraries were then pooled and enriched for HBV-containing reads using custom-designed xGen Lockdown probes (Integrated DNA Technology) of 60 bp each tiling the entire HBV genome and oligo capture protocol was followed per manufacture’s protocol. Enriched libraries were subsequently amplified using 11 PCR cycles with NEBNext High-Fidelity 2X PCR Mix and purified using Ampure XP beads.
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| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
NextSeq 1000 |
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| Description |
MNase-seq
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| Data processing |
UMI demultiplexing (custom script)
adapter trimming (custom script)
alignment to HBV genome (Bowtie2 v2.3.4.3)
nucleosome coverage mapping (nucleoATAC)
Bedgraphs from both RNA- and MNase-seq were plotted using Sushi package v1.30.0 in R v4.1.0
Assembly: NC_003977.2 (offset by 500 bp)
Supplementary files format and content: bedgraph files that include coverage of fragment centers (50 bp windows)
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| Submission date |
Feb 21, 2023 |
| Last update date |
Mar 03, 2023 |
| Contact name |
Andres Mansisidor |
| E-mail(s) |
mansisidor@nyu.edu, mansisidor@rockefeller.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 |
GPL30882 |
| Series (2) |
| GSE225715 |
A nucleosome switch primes Hepatitis B Virus infection [Mnase-Seq] |
| GSE225716 |
A nucleosome switch primes Hepatitis B Virus infection |
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| Relations |
| BioSample |
SAMN33399641 |
| SRA |
SRX19461923 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7054615_UMI_dedup_HBV_00hr_0250U_MNase_TCGGATTC_TAGTTGCG_S00_001.trim.st.all.qft_50bpWindow.cov.bedgraph.gz |
419 b |
(ftp)(http) |
BEDGRAPH |
| GSM7054615_UMI_dedup_HBV_00hr_0250U_MNase_TCGGATTC_TAGTTGCG_S00_001.trim.st.all.qft_50bpWindow.cov.bedgraph.gz.tbi.gz |
243 b |
(ftp)(http) |
TBI |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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