|
| Status |
Public on Feb 15, 2022 |
| Title |
HSB2.dRNAseq.mock.B |
| Sample type |
SRA |
| |
|
| Source name |
HSB2 cells (HHV-6 Foundation, USA)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HSB2 cells (HHV-6 Foundation, USA)
cell type: Childhood T acute lymphoblastic leukemia cells
infection: No infection; Replicate 2
fraction: Total RNA used for dRNA-seq
|
| Treatment protocol |
HSB2 cells were infected with HHV-6A at an mOI of 5-10
|
| Growth protocol |
HSB2 cells were grown in RPMI1640 media in presence of Streptomycin and Penicilin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using TRI-reagent (SIGMA) following manufacturer's instructions.
Custom
HSB-2 cells were obtained from HHV-6 Foundation, USA and were maintained in RPMI 1640 media with 200 units of Penicilin and Streptomycin. HSB2 cells were infected with HHV-6A atan MOI of 5-10. 3 days post infection, total RNA was prepared using TRI-reagent (SIGMA). Two biological replicates were prepared on two different days. Transcription start site profiling dataset using dRNA-seq was prepared according to the published protocol (Sharma and Vogel, 2014) with some modifications by the Core Unit Systems Medicine (Würzburg). In brief, total RNA was extracted from HHV-6A and mock infected HSB-2 cells. For each sample 3 µg of DNase-digested RNA was treated with T4 Polynucleotide Kinase (NEB) for 1 h at 37 °C. RNA was purified with Oligo Clean & Concentrator columns (Zymo) and each sample was split into an Xrn1 (+Xrn1) and a mock (−Xrn1) sample. The samples were treated with 1.5 U Xrn1 (NEB; +Xrn1) or water (−Xrn1) for 1 h at 37 °C. Digest efficiency was checked on a 2100 Bioanalyzer (Agilent) and 5′ caps were removed by incubation with 20 U of RppH (NEB) for 1 h at 37 °C. Afterwards, RNA was purified and eluted in 7 µL and 6 µL were used as input material for the NEBNext Multiplex Small RNA Library Prep Set for Illumina. Library preparation was performed according to the manufacturer’s instruction with the following modifications: 3′ adapter, SR RT primer and 5′ adapter were diluted 1:2, 13 cycles of PCR were performed with 30 sec of elongation time, and no size selection was performed at the end of library preparation. Concentrations of libraries were determined using the Qubit 3.0 (Thermo Scientific) and their fragment sizes were determined using the Bioanalyzer. Libraries were pooled equimolar. Sequencing of 75 bp single-end reads was performed on a NextSeq 500 (Illumina) at the Core Unit Systems Medicine in Würzburg (dRNA-seq).
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Reads that passed the chastity filter on the NextSeq 500 were subject to de-multiplexing and trimming of TriLink adapter residuals using Illumina’s bcl2fastq v2 software (v2.19.1). To refine the output, read quality was checked with FastQC software (v0.11.5) and reads shorter 10 bases or longer 25 bases or containing “N” as a base call, were discarded.
STAR_2.5.3a was used to map all reads against a combined index prepared for the human genome (Ensembl 90) and the HHV6A genome (genbank accession X83413). The following parameters were used: --alignIntronMax 1 --outFilterMismatchNmax 1 --outFilterMultimapScoreRange 0 --outFilterMultimapNmax 10 --outFilterScoreMinOverLread 0 --outFilterMatchNminOverLread 0 --outFilterMatchNmin 16.
TSS were called with iTiSS version 1.2
Details of dRNA sequencing data analysis is previously published by us (Whisnant et al., 2020).
Genome_build: hg38, X83413
|
| |
|
| Submission date |
Jul 10, 2021 |
| Last update date |
Feb 15, 2022 |
| Contact name |
Florian Erhard |
| E-mail(s) |
Florian.Erhard@uni-wuerzburg.de
|
| Organization name |
Julius-Maximilians-Universität Würzburg
|
| Department |
Institut für Virologie und Immunbiologie
|
| Street address |
Versbacher Str. 7
|
| City |
Würzburg |
| ZIP/Postal code |
97078 |
| Country |
Germany |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE179866 |
A herpesvirus microRNA targets host microRNA processing to promote virus reactivation from latency [dRNA-seq] |
| GSE179867 |
A herpesvirus microRNA targets host microRNA processing to promote virus reactivation from latency |
|
| Relations |
| BioSample |
SAMN20164767 |
| SRA |
SRX11406252 |
