 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Apr 01, 2022 |
| Title |
WT R6 field rep3 |
| Sample type |
SRA |
| |
|
| Source name |
seed
|
| Organism |
Glycine max |
| Characteristics |
genotype: Thorne (WT)
growth condition: field
time point: R6
tissue: seed
|
| Growth protocol |
Field Condition: Field trials were conducted on homozygous transgenic events and lineages of T4, and T5 generations of transgenic plants, WD-1 and WD-2, in crop seasons 2019. All evaluations were conducted in completely randomized plots with three replications. Each plot consisted of four 10 ft rows in a four-row plot design in which two border rows were the wildtype (WT) Thorne. One hundred seeds were sown in each row. Irrigation was provided when necessary using a sprinkler system. Seeds were collected at R5, R5/6, and R6 from three replications. A small portion of twelve seeds from each sample were used for genotyping for the presence of the transgenes and the rest portion of the seeds were bulked to extract RNA. Greenhouse condition: The seeds were planted in 30 by 26 cm plastic pots (Nursery supplies Inc., Orange, CA, USA) containing Baccto, Horticultural sphagnum peat and perlite (Michigan peat company, Houston, TX, USA). After planting, the pots were placed in a greenhouse maintained at 30° and 25° C, day and night, respectively, with a photo-period of 16 h. The light intensity varied but was, on average, approximately 450 μmol.m–2.s–1. The three genotypes, WD-1, WD-2 and the control WT, were potted every two weeks (four pots each). The plants were separated in the greenhouse using pollen screens to avoid cross-pollination between genotypes. After the first 45 days, the plants were fertilized every 2 weeks with 6 g of 15-5-15 fertilizer (Everris NA Inc., Dublin, OH, USA). Once the plants start flowering, each day the flowers that open were tagged in order to identify the correct developmental stages. Samples for biomass, metabolomics and transcriptomic analysis were collected at different points of development: 21, 24, 27, 30, 33, 36 days after flowering (DAF) and at maturity. The collected immature seeds were peeled, removing the seed coat and endosperm and then were frozen under liquid N2. Each replicate consisted in one embryo per tube, except for 21 and 24 DAF, where three embryos were used and at least four replicates were collected for each time point and line. For the mature stage, embryos were ground for each replicate and 4 replicates were analyzed. Finally, all the samples were freeze dried in a liophilator for 3 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Regarding field samples, twelve seeds from each pot sample were genotyped and four seeds were bulked for RNA extraction using the QIAGEN RNAEseay Kit (QIAGEN, INC.). The purified RNA was checked for quality on 1% agarose gel. Concentration was quantified using the Nanodrop. The RNA was cleaned from any DNA contamination by DNAse treatment using the Turbo DNA free kit (Invitrogen, INC.). Regarding greenhouse samples, immature embryos of the same green-house grown plants as the metabolomics analysis were used to collect RNA for the transcriptomics analysis. For each of the three genotypes, RNA was extracted at four timepoints of 24, 27, 30, and 33 DAP and from three plants as three replicates. For each replicate, a single embryo was flash-frozen with liquid nitrogen and ground with pestle and mortar into fine powder from which RNA was isolated and cleaned using the Macherey-Nagel NucleoSpin RNA Plant Mini kit (MACHEREY-NAGEL Inc.).
RNA libraries were prepared for sequencing using standard Illumina protocols
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Short reads were assessed for quality control using the FastQC program (version 0.11.5).
High-quality reads were loaded to kallisto (version 0.43.1) available as a graphic user interface on Cyverse (www.cyverse.org) with bootstrap value set at 100 and k-mer length at 31. AtWRI1 and AtDGAT sequences used in the transformation vector were added to the G. max cDNA database corresponding to the Wm82.a2.v1 genome retrieved from Ensembl (http://plants.ensembl.org). This cDNA library served as the transcriptome reference for pseudo-alignment and read quantification.
Results from kallisto were used as input for differential expression analysis at gene level via the sleuth package (version 0.30.0) in R (version 3.6.3).
Raw count files (.csv) were generated via extracting information in the kallisto "abundance.tsv" file of each sample into a matrix in R, matching transcript IDs with gene IDs, and summing estimated counts (marked as "est_counts") of all transcript of the same gene.
Genome_build: Glycine max [L.] Merr Wm82.a2.v1
Supplementary_files_format_and_content: Tab-delimited .csv files include raw gene counts for each sample
|
| |
|
| Submission date |
Oct 18, 2021 |
| Last update date |
Apr 02, 2022 |
| Contact name |
Tu Huynh |
| E-mail(s) |
[email protected]
|
| Phone |
(832) 980-4243
|
| Organization name |
Ohio State University
|
| Department |
Horticulture and Crop Science
|
| Street address |
2021 Coffey Rd
|
| City |
Columbus |
| State/province |
OH |
| ZIP/Postal code |
43210 |
| Country |
USA |
| |
|
| Platform ID |
GPL28801 |
| Series (1) |
| GSE186058 |
Expression of Arabidopsis WRI1 and DGAT1 during soybean embryo development alters oil and carbohydrate composition |
|
| Relations |
| BioSample |
SAMN22371144 |
| SRA |
SRX12671781 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5629938_C1-3.csv.gz |
283.6 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
