|
| Status |
Public on Jan 05, 2022 |
| Title |
R-6-30-1 |
| Sample type |
SRA |
| |
|
| Source name |
HeP-2 cells, S2 Cells, HSV-13
|
| Organisms |
Drosophila melanogaster; Homo sapiens; Human alphaherpesvirus 1 |
| Characteristics |
cell line (human): HeP-2 cells
cell line (drosophila): S2 cells
cell type (human): lung epithelial carcinoma cells
viral infection: infected with HSV-1 US1/US1.5 deletion virus with repaired US1/US1.5 gene
infection timepoint: 6 hours post infection
run-on time: 30 min of run on with Br-UTP
|
| Treatment protocol |
Hep-2 cells were infected with the mutant viruses at a MOI of 5 and allowed to progress to 3 or 6 hours post infection.
|
| Growth protocol |
Hep-2 cells were grown and maintained with DMEM 10% fetal bovine serum and the Drosophila S2 cells were grown in Schneider's medium supplemented with 10% NBS
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Nuclei were extracted as previoulsy described (Birkenheuer and Baines 2020, Journal of Virology). Global run-on reactions were performed as described by Lopes et al 2017, GRO-seq, A Tool for Identification of Transcripts Regulating Gene Expression. Methods Mol Biol 1543:45-55, and were allowed to proceed for 5 or 30 mins of run-on. Reactions were stopped by the addition of Trizol.
Libraries were constructed similar to the construction of PRO-seq libraries with adapter ligations for the 3' end being performed first and cap removal and 5' phosphate repair and 5' adapter ligation being performed after the 3' end of the RNA was ligated to the sequencing adapter. However, instead of using magnetic beads for this process, anti Br-dUTP conjugated agarose beads were used for this process with the blocking, washing, and elution conditions described by (Lopes et al, 2017, GRO-seq, A Tool for Identification of Transcripts Regulating Gene Expression. Methods Mol Biol 1543:45-55.).
Libraries were created using Illumina Index primers for sequencing on the NextSeq550 75 base pair single end sequencing.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
repair-infected HeP2 cells at 6 hpi with 30 min of run on rep 1
labeled RNA was extracted and prepared for sequencing with sequential, specific 3' and 5' RNA adapters. Libraries were reverse transcribed and sequenced with Illumina Next Seq550 75bp single end sequencing, the spiked in drosophila s2 cells were used for normalization after BigWig creation
|
| Data processing |
Library strategy: PRO-seq
Adapter trimming and processing using Cut Adapt
Raw genome algnments usign BWA
Conversion of BAM files to strand specific BigWigs using kentsource
https://github.com/Danko-Lab/tutorials/blob/master/PRO-seq.md
Genome_build: hg38 human genome build, Dm3 Drosophila genome build, HSV-1 (F) strain in-house genome build
Supplementary_files_format_and_content: read count data in BigWig files
|
| |
|
| Submission date |
Dec 07, 2021 |
| Last update date |
Jan 05, 2022 |
| Contact name |
Joel D Baines |
| E-mail(s) |
[email protected]
|
| Organization name |
Cornell University
|
| Street address |
235 Hungerfordhill Rd
|
| City |
Ithaca |
| State/province |
NY |
| ZIP/Postal code |
14853 |
| Country |
USA |
| |
|
| Platform ID |
GPL29914 |
| Series (1) |
| GSE169574 |
ICP22 of Herpes Simplex Virus 1 decreases RNA Polymerase Processivity |
|
| Relations |
| BioSample |
SAMN23722879 |
| SRA |
SRX13340051 |
