|
| Status |
Public on Mar 05, 2021 |
| Title |
Pol2_24hpi_Rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Pol2_24hpi GFP+
|
| Organisms |
Homo sapiens; Human betaherpesvirus 5 |
| Characteristics |
treatment: live GFP+ infected, 24hpi
chip antibody: Rpb1 NTD (D8L4Y) Rabbit mAb #14958 (Cell Signaling Technologies)
cell line: Kasumi-3
|
| Treatment protocol |
Kasumi-3 were infected with TB40/E wtGFP strain of HCMV at MOI 1 or mock-infected. Extracellular virus was inactivated at 24 hours post infection. HCMV-infected cells were sorted for GFP expression and live cells. Mock-infected cells were sorted for live cells. Cells were then fixed with 1% of Formaldehyde in PBS fand quenched with 0.125 M glycine.
|
| Growth protocol |
Kasumi-3 were grown in RPMI-1640 medium (ATCC 30-2001) supplemented with 20% fetal bovine serum (Corning) and 1,000 Units/ml Penicillin-Streptomycin (Gibco)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
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 (D5E4) XP Rabbit mAb #8173 (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 (Bio 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).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Adapters were trimmed from reads using cutadapt version 1.13
Trimmed reads were aligned to the reference genome using Bowtie2 version 2.2.6
bigWig files were created using HOMER
Genome_build: hg19
Genome_build: HCMV EF999921.1 [HCMV genome fa and gtf files for alignment of bigwig files are available at https://github.com/Mary-Hummel/HCMV-TB40-E-GFP-reference-genome.git]
Supplementary_files_format_and_content: bigWig files
|
| |
|
| Submission date |
Jan 14, 2021 |
| Last update date |
Mar 05, 2021 |
| Contact name |
Mary Hummel |
| E-mail(s) |
m-hummel@northwestern.edu
|
| Phone |
847-322-6438
|
| Organization name |
Northwestern University
|
| Street address |
303 E. Chicago Ave
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60611 |
| Country |
USA |
| |
|
| Platform ID |
GPL24958 |
| Series (2) |
| GSE164845 |
Pol2 occupancy of the viral and host genomes in HCMV infected Kasumi-3 cells at 24 hpi |
| GSE164861 |
Epigenetic reprogramming of host and viral genes by Human Cytomegalovirus infection in Kasumi-3 myeloid progenitor cells at early times post-infection |
|
| Relations |
| BioSample |
SAMN17315709 |
| SRA |
SRX9850048 |
