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| Status |
Public on Dec 04, 2019 |
| Title |
Noff_stem_air_1d_R3 |
| Sample type |
SRA |
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| Source name |
stems of 3-week-old plants
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| Organism |
Nasturtium officinale |
| Characteristics |
treatment: air
treatment duration: 1 day
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| Treatment protocol |
Plants of the 5-6 leaf stage were selected for developmental homogeneity before the start of the experiments. Two hours after the start of the photoperiod, plants were completely submerged under short-day conditions in plastic tubs, filled with temperature-adjusted water, or kept in air in similar plastic tubs. Stems and petioles of air and submerged plants (five plants pooled per sample) were harvested separately after 1 and 2 days and frozen in liquid nitrogen.
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| Growth protocol |
For germination, several seeds were sown in the same pot on sterilized soil. After 6-7 d, germinated seedlings were transplanted to single pots. Plants grew under short-day conditions (8 h light/ 16 h dark cycle), 23 °C, and 100 μmol photons m-2 s-1 for about two more weeks.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen samples of three replicates were ground for 3 min in 2 ml tubes with Zirconia/Silica beads using Mini-Beadbeater (BioSpec Products, Bartlesville, OK, USA) in plates frozen with liquid nitrogen. After grinding, 1 ml Lysis binding buffer (Dynabeads mRNA Direct Kit, Thermo Fisher Scientific, Waltham, MA, USA) was added and samples were homogenized with this buffer for an additional 2 min in the Mini-Beadbeater. Homogenized samples were centrifuged at 10,000 rpm for 3 min and loaded onto a homogenizer spin column (Omega Bio-Tek, Norcross, GA, USA) and centrifuged to remove excess tissue. Lysates were transferred to a 96-well plate and stored at -80°C until library preparations.
RNA-seq libraries were constructed using BrAD-seq protocol for non-strand specific libraries (Townsley et al., 2015). 200 μl of lysates were used for mRNA isolation, following manufacturer’s instructions (Dynabeads mRNA Direct Kit, Thermo Fisher Scientific, Waltham, MA, USA). Isolated mRNA was fragmented by RT buffer for 90 sec at 94°C (RevertAid RT enzyme, Thermo Fisher Scientific, Waltham, MA, USA), and cDNA synthesis was done on the fragmented mRNA. Second strand synthesis was done according to BrAD-seq protocol and was followed by adapter ligation. PCR enrichment was performed using unique indexed oligos for multiplexing. After purification, sample concentration was measured on a plate reader (with SYBR Green I Nucleic Acid Gel Stain, Invitrogen, Carlsbad, CA, USA) and sample concentrations for multiplexing were adjusted accordingly. Multiplexing within the plate was done in a completely randomized design. Multiplexed samples were analyzed on a Bioanalyzer for contamination and size confirmation. The size of the fragments varied between 200 and 500 bps. Samples were sequenced on Hiseq4000 with 100 bp paired-end mode.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Adapters were trimmed from the FASTQ files using the BBDuk algorithm.
Quantification of aligned reads to the published transcriptome by Voutsina et al., 2016 was performed using Kallisto (Bray et al., 2016).
Differential expression analysis was performed with the edgeR and limma Bioconductor packages in R. Log2 Fold Changes and Benjamini-Hochberg-corrected P-values were calculated fitting a negative binomial generalized log-linear model with a full factorial design matrix of treatment and tissue.
Genome_build: PRJNA284126
Supplementary_files_format_and_content: tab-separated text files include length of transcript, est_counts and tpm for each sample
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| Submission date |
Sep 25, 2019 |
| Last update date |
Dec 05, 2019 |
| Contact name |
Angelika Mustroph |
| E-mail(s) |
[email protected]
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| Organization name |
University Bayreuth
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| Department |
Plant Physiology
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| Lab |
Angelika Mustroph
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| Street address |
Universitaetsstr. 30, NWI
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| City |
Bayreuth |
| ZIP/Postal code |
95440 |
| Country |
Germany |
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| Platform ID |
GPL27529 |
| Series (1) |
| GSE138020 |
Keeping the shoot above water – underwater stem elongation in watercress (Nasturtium officinale) |
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| Relations |
| BioSample |
SAMN12843088 |
| SRA |
SRX6902487 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4096761_abundance_A_III_2SV4.tsv.gz |
1.0 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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