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| Status |
Public on Feb 15, 2022 |
| Title |
mir.HHV6A.C (lytic) |
| Sample type |
SRA |
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| Source name |
HSB2 cells (HHV-6 Foundation, USA)
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HSB2 cells (HHV-6 Foundation, USA)
cell type: Childhood T acute lymphoblastic leukemia cells
infection: HHV-6A infection; Replicate 3
fraction: Total RNA used for small RNA-seq
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| Treatment protocol |
HSB2 cells were infected with HHV-6A at an mOI of 5-10
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| Growth protocol |
HSB2 cells were grown in RPMI1640 media in presence of Streptomycin and Penicilin.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using TRI-reagent (SIGMA) following manufacturer's instructions.
NEXTflex Small RNA-seq kit v3 (Bioo Scientific, Germany)
HSB-2 cells were obtained from HHV-6 Foundation, USA and were maintained in RPMI 1640 media. For HHV-6A infecttion, cells were seeded into 6-well plates and were infected with cell free virus at a MOI of 5-10. Virus infection was allowed to proceed for 72 h. Afterwards, cells were washed once with PBS and total RNA was extracted using TRI reagent. To study Argonaut bound miRNAs, Ago2 immunoprecipitation was carried out using a previously published protocol (Hauptmann et al., 2015) with minor modifications. 5 million HSB-2 cells were infected with HHV-6A for 3 days as described above. Afterwards, native protein lysates were prepared from mock infected and HHV-6A infected cells using lysis buffer (50 mM Hepes, pH 7,5; 150 mM NaCl; 0.5% NP-40; 1 mM NaF; 10% Glycerol, 2.5 mM MgCl2; protease inhibitor (Roche), 0.5 mM DTT; 10 units/ml RNAse inhibitor (NEB, M0314L)). Anti-flag magnetic beads were washed with PBS and were incubated with TNR6B-flag peptide (Hauptmann et al., 2015) for overnight at 4 C on a head over tail rotor. Next day, beads were washed once with PBS and once with lysis buffer. Afterwards half of the beads were incubated with the protein lysate for overnight at 4C on a head over tail rotor. Rest of the beads were stored at 4 degree C. Next day, beads were separated from the lysate on a magnet. Collected protein lysate was then mixed with remaining TNR6B-flag beads and were allowed to incubate for another overnight at 4 degree C on a head over tail rotor for further protein-antibody binding. Collected beads were washed 4 times with wash buffer (50 mM Hepes, pH 7,5; 150 mM NaCl; 1 mM NaF; 10% Glycerol, 2.5 mM MgCl2; protease inhibitor (Roche), 0.5 mM DTT; 10 units/ml RNAse inhibitor (NEB, M0314L)). Finally, beads were resuspended in PBS containing 0.5 mM MgCl2. A small fraction of these beads were used for denatured protein lysate preparation (eluate 1) for immunoblotting. Remaining beads were lysed in TRI-reagent and RNA was extracted. Next day, re-incubated beads were processed as described above to get the eluate 2 and the second batch of Ago2-bound RNA. Total RNA from both the batches were pooled together and processed for miRNA sequencing. miRNA sequencing was carried out at CeGaT GmbH, Tübingen usint NEXTflex Small RNA-seq kit v3 (Bioo Scientific, Germany).
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| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
HSB2.counts.tsv.gz
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| 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.
Genome_build: hg38, X83413
Supplementary_files_format_and_content: HSB2.counts.tsv.gz
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| Submission date |
Jul 10, 2021 |
| Last update date |
Feb 15, 2022 |
| Contact name |
Florian Erhard |
| E-mail(s) |
Florian.Erhard@uni-wuerzburg.de
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| Organization name |
Julius-Maximilians-Universität Würzburg
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| Department |
Institut für Virologie und Immunbiologie
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| Street address |
Versbacher Str. 7
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| City |
Würzburg |
| ZIP/Postal code |
97078 |
| Country |
Germany |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE179865 |
A herpesvirus microRNA targets host microRNA processing to promote virus reactivation from latency [Lytic infection] |
| GSE179867 |
A herpesvirus microRNA targets host microRNA processing to promote virus reactivation from latency |
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| Relations |
| BioSample |
SAMN20164759 |
| SRA |
SRX11406246 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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