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| Status |
Public on Mar 05, 2021 |
| Title |
Pol2 occupancy of the viral and host genomes in HCMV infected Kasumi-3 cells at 24 hpi |
| Organisms |
Homo sapiens; Human betaherpesvirus 5 |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
Purpose: to compare Pol2 occupancy of the host genome in HCMV infected and mock infected Kasumi-3 cells at 24 hours post infection and analyze Pol2 occupancy of the viral genome in GFP+ infected cells
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| Overall design |
Kasumi-3 cells were infected with the TB40/E wtGFP strain of HCMV. At 4 hr post-infection, virus was inactivated and cells left in culture for additional 20 hours. Infected cells were sorted for GFP expression and live cells. Mock infected cells were sorted for live cells. After fixation, cells were lysed and sonication with Covaris Focused-ultrasonicator at 10% Duty Cycle, 140 peak, 200 cycles per burst, 360 seconds. ChIP was performed by incubating the sonicated sample with Rpb1 NTD (D8L4Y) Rabbit mAb #14958 (Cell Signaling Technologies) and the protein A/G agarose beads. Beads were washed and eluted in 200μl of elution buffer (50 mM Tris-HCl at pH 8.0, 10 mM EDTA, 1.0% SDS). The eluates were de-cross-linked and the DNA purified using the Qiagen PCR purification kit. Libraries were prepared with the KAPA HTP Library Preparation Kit multiplexed with NEXTflex DNA Barcodes (Bioo Scientific) and size selected with AMPure XB beads (Beckman Coulter) to have fragments 250-450 bp in length. Library quality control was assessed using Bioanalyzer High Sensitivity DNA Analysis kit (Agilent) and sequencing was performed with Illumina NextSeq technology (50bp single- end reads).
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| Contributor(s) |
Hummel M, Forte E, Schipma M |
| Citation(s) |
33731453 |
| NIH grant(s) |
| Grant ID |
Grant title |
Affiliation |
Name |
| P01 AI112522 |
Integrating Mechanistic Insights from Diverse Models to Prevent CMV Reactivation following Transplantation |
NORTHWESTERN UNIVERSITY AT CHICAGO |
MARY A HUMMEL |
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| Submission date |
Jan 14, 2021 |
| Last update date |
Jun 04, 2021 |
| Contact name |
Mary Hummel |
| E-mail(s) |
m-hummel@northwestern.edu
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| Phone |
847-322-6438
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| Organization name |
Northwestern University
|
| Street address |
303 E. Chicago Ave
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| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60611 |
| Country |
USA |
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| Platforms (3) |
| GPL18573 |
Illumina NextSeq 500 (Homo sapiens) |
| GPL20301 |
Illumina HiSeq 4000 (Homo sapiens) |
| GPL24958 |
Illumina NextSeq 500 (Homo sapiens; Human betaherpesvirus 5) |
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| Samples (8)
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| This SubSeries is part of SuperSeries: |
| GSE164861 |
Epigenetic reprogramming of host and viral genes by Human Cytomegalovirus infection in Kasumi-3 myeloid progenitor cells at early times post-infection |
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| Relations |
| BioProject |
PRJNA692197 |
| SRA |
SRP301770 |
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