|
| Status |
Public on Mar 05, 2020 |
| Title |
TB40 72h IE2F 6h PFA |
| Sample type |
SRA |
| |
|
| Source name |
HCMV-infected primary human foreskin fibroblast
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: primary human foreskin fibroblast
hcmv strain: TB40 BAC4
infection duration: 72 hours
sequencing method: PRO-Seq
|
| Treatment protocol |
HFF were infected with the indicated virus at an MOI of 1-3 for the indicated duration. Cells were treated with the indicated drugs (dTAG, Flavo, and/or PFA) or appropriate vehicle controls for the indicated times during the final hours of infection before harvest.
|
| Growth protocol |
HFF were isolated and cultured as described in the manuscript. Cells were maintained in Minimum Essential Medium and grown to confluence for at least one week before infection. All HFF used for these studies were at passage number ≤ 6.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Nuclei isolation was carried out as described in Ball et. al. (2019, PMID: 30743000) and Parida et. al. (2019, PMID: 30755505).
PRO-Seq library preparation to extract and sequence nascent RNAs is carefully detailed in the manuscript and was carried out as previously described by Parida et. al. (2019, PMID: 30755505), with noted modifications.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
nascent RNA with moth spike-in cells
|
| Data processing |
Library strategy: PRO-Seq
Adapter trimming using trim_galore 0.4.4.
Alignment of trimmed reads to concatenated hg38 (human), FJ616285.1 or KF297339.1 (Towne and TB40, respectively), and, for samples that included moth spike-in cells, JQCY02.1 (spodoptera) genome assemblies.
Deduplication of mapped reads according to unique molecular identifiers using dedup (https://github.com/P-TEFb/dedup).
Reads were parsed out based on the genome to which they aligned.
Bedgraphs were generated using bedTools genomecov 2.26 and data were normalized by library size and spike-in reads accorind to Ball et. al. (2019, PMID: 30743000).
Bigwigs were generated using kentUtils
hg38 bigwigs were uploaded to the UCSC genome browser.
Custom UCSC track hubs were set up for Towne and TB40 data.
For analysis of host transcription effects upon infection or depletion of IE2 and annotation of gene 5' ends based on PRO-Seq data, we applied a new truQuant algorithm that utilizes our previously published tsrfinder tool. Our application of these tools is described in the manuscript. truQuant source code is avaliable on GitHub at https://github.com/meierjl/hubs/tree/master/truQuant.
Genome_build: hg38 (human), FJ616285.1 (HCMV Towne, KF297339.1 (HCMV TB40), JQCY02.1 (Spodoptera).
Supplementary_files_format_and_content: bigwigs contain PRO-Seq pileups. BED file contains an annotation of human gene bodies queried for effects on transcription.
|
| |
|
| Submission date |
Oct 18, 2019 |
| Last update date |
Mar 05, 2020 |
| Contact name |
David H Price |
| E-mail(s) |
david-price@uiowa.edu
|
| Organization name |
University of Iowa
|
| Department |
Department of Biochemistry and Molecular Biology
|
| Street address |
431 Newton Road
|
| City |
Iowa City |
| State/province |
IA |
| ZIP/Postal code |
52242 |
| Country |
USA |
| |
|
| Platform ID |
GPL20301 |
| Series (1) |
| GSE139114 |
Human cytomegalovirus IE2 drives transcription initiation from a select subset of late infection viral promoters by host RNA polymerase II |
|
| Relations |
| BioSample |
SAMN13060803 |
| SRA |
SRX7023979 |
