|
| Status |
Public on Feb 15, 2022 |
| Title |
U2OS.-HHV6A.-TSA.A (latent) |
| Sample type |
SRA |
| |
|
| Source name |
U2-OS
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: U2-OS
cell type: Human Osteosarcoma cells carrying no HHV-6A
time: 48 h p.i.
treatment: DMSO (solvent control)
|
| Treatment protocol |
Cells were washed with PBS just before the treatment and then were replaced with freash media containing Ethanol/DMSO/Doxycycline/TSA.
|
| Growth protocol |
Cells were grown in RPMI1640 media with 10% FCS without any antibiotics.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were washed once with PBS and total cellular RNA was extracted using Qiagen RNeasy mini kit.
RNA-seq libraries were prepared with the TruSeq Stranded mRNA LT sample preparation kit (Illumina) using both, Sciclone and Zephyr liquid handling robotics (PerkinElmer). Library concentrations were quantified with the Qubit 2.0 Fluorometric Quantitation system (Life Technologies) and the size distribution was assessed using the Experion Automated Electrophoresis System (Bio-Rad). For sequencing, samples were diluted and pooled into NGS libraries in equimolar amounts and were sequenced using NextSeq 500.
U2-OS cells were purchased from ATCC and were cultured in McCoy5A medium supplemented with 10% (v/v) FBS and 200 units/ml penicillin-streptomycin. U2-OS cells ccarrying latent HHV-6A were reactivated by treating the cells with 80 ng/ml of TSA for 48 h. To generate small RNA sequencing libraries, total cellular RNA was isolated using Qiagen miRNeasy kit and checked for signs of RNA degradation on a 2100 Bioanalyzer instrument (Agilent). Small RNA stranded libraries were prepared using the CleanTag Ligation Kit (TriLink BioTechnologies) starting with 1µg of total RNA per library. For quality control, all final libraries were analyzed by Bioanalyzer to check for the expected miRNA fragment peak at 141 bp. Subsequently, a Pippin Prep instrument (Sage Science) was used to isolated fragments between 10 and 35 bp in size. Library concentrations were measured by PicoGreen on an infinite F200 instrument (Tecan). Libraries were then sequenced on a NextSeq 500 platform (Illumina) using high-output v2 kits with 75 cycles aiming for about 40 million single-end reads per sample.
|
| |
|
| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
small RNA between 10-25 nt length
U2OS.counts.tsv.gz
|
| 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.
Reads were counted using Gedi 1.0.3.
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). Read quality was checked with FastQC software (v0.11.5). Reads shorter than 18 bases were discarded. All reads were mapped to combined index of the human genome (HG38) and the HHV-6A genome (X83413), with the trailing repeat region masked by N. Read mapping was performed using STAR (v2.5.3a) using the parameters suggested by the Encode project for sRNA-seq. Reads were then annotated to mature miRNAs if 5’ end perfectly matched (miRBase 22.1). The raw counts were then normalized and analyzed for differential miRNA expression with the DESeq2 (v1.18.1) package. Statistically significantly differentially regulated genes were called by a Wald test for the interaction of the presence of HHV-6A (or the sncRNA) and activation by TPA (or Dox) with FDR 1% (Benjamini-Hochberg corrected p-value).
Genome_build: hg38, X83413
Supplementary_files_format_and_content: U2OS.counts.tsv.gz
|
| |
|
| 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) |
| GSE179864 |
A herpesvirus microRNA targets host microRNA processing to promote virus reactivation from latency [Latent reactivation] |
| GSE179867 |
A herpesvirus microRNA targets host microRNA processing to promote virus reactivation from latency |
|
| Relations |
| BioSample |
SAMN20164750 |
| SRA |
SRX11406237 |
