|
| Status |
Public on Jun 08, 2021 |
| Title |
EV-4 (Mock-treated EVs, biological replicate 4) |
| Sample type |
SRA |
| |
|
| Source name |
AB33 induced filament EVs
|
| Organism |
Ustilago maydis |
| Characteristics |
strain: AB33
sample type: EVs
treatment: Mock (PBS)
|
| Treatment protocol |
For each replicate, EVs from approximately 300ml of culture supernatant were treated with either 0.1 µg/µl RNase A in phosphate buffered saline (PBS) or just PBS for mock treatment at 4 degrees Celsius for 10 minutes.
|
| Growth protocol |
Initial sporidial cultures of strain AB33 from Brachmann et al. (2001) were grown to OD600 1.0 ± 0.1 in complete medium according to Holliday (1974), supplemented with 1% glucose (w/v). The cells were shifted to nitrate minimal medium with 2% glucose to induce filamentation. All cultures were grown at 28 degrees Celsius with shaking at 200 rpm. Cells were harvested and EVs isolated from the culture supernatant at 15-16 hours post induction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells and EVs were snap frozen and stored at -80 degrees Celsius until extraction. RNA was extracted from EVs and filament samples using standard methods for TRI-reagen LS (Sigma) and TRI-reagent (Sigma), respectively. Extracted RNA was treated with DNase I (ThermoFisher) and re-extracted with TRI-reagent LS. Coprecipitant GlycoBlueTM (ThermoFisher) was used for EV RNA samples for the first extraction and for all samples in the re-extraction.
Libraries for sequencing were generated with NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina together with NEBNext® Poly(A) mRNA Magnetic Isolation Module according to the manufacturer's instructions.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
| |
|
| Data processing |
Base-calling with Illumina RTA 1.18.54.4
Paired-end merging with BBMerge (v 2019) mininsert=24 mininsert0=24 minoverlap=6 minoverlap0=6 ecct extend2=20 iterations=5
Alignment to reference with Hisat2 (v 2.1.0) --dta --no-softclip --known-splicesite-infile
Read count correction for degraded samples with DegNorm (v 0.1.4) default
differential expression analysis using the DegNorm corrected counts with DESeq2 (v 1.32.0) lfcShrink
Genome_build: Umaydis521_2.0, ENSEMBL
Supplementary_files_format_and_content: DegNorm corrected count matrix CSV file, columns = samples, rows = genes
Supplementary_files_format_and_content: DESeq2 results CSV file, rows = genes, columns = see header
|
| |
|
| Submission date |
Jun 07, 2021 |
| Last update date |
Jun 08, 2021 |
| Contact name |
Michael Feldbrügge |
| E-mail(s) |
feldbrue@hhu.de
|
| Phone |
+49 211 8115475
|
| Organization name |
Heinrich-Heine University Düsseldorf
|
| Department |
Biology
|
| Street address |
Universtitätstr. 1
|
| City |
Düsseldorf |
| ZIP/Postal code |
40225 |
| Country |
Germany |
| |
|
| Platform ID |
GPL30238 |
| Series (1) |
| GSE176292 |
Comparison of extracellular vesicle and filamentous cell transcriptomes of Ustilago maydis |
|
| Relations |
| BioSample |
SAMN19594790 |
| SRA |
SRX11083714 |
