|
| Status |
Public on Mar 14, 2019 |
| Title |
EV_60N_Rep1 |
| Sample type |
SRA |
| |
|
| Source name |
root protoplasts
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Col-0
tissue: root protoplasts
expression vector: empty
treatments: 60N, Cycloheximide, Dexamethasone
age: 10 days
|
| Treatment protocol |
Protoplasts were isolated from root tissue and transfected with expression vector. The following morning protoplasts were treated with the indicated N supply (0N = 20 mM KCL, 5N = 5 mM KNO3, and 60N = 20 mM KNO3 + 20 mM NH4NO3), +/- 35 uM cycloheximide and 10 uM dexamethasone. Whole roots were treated with 20 mM KCl or 20 mM KNO3 + 20 mM NH4NO3.
|
| Growth protocol |
Arabidopsis seedlings grown in MS basal media w/o C or N + 1% sucrose and 1mM KNO3. For protoplast isolation, plants were grown on vertical plates and for whole root N-response plants were grown in liquid media in Phytatrays.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For protoplast samples, approximately 10,000-20,000 cells were FACS sorted into RLT buffer and frozen. For whole tissue, roots were transferred into microfuge tubes and flash frozen in liquid nitrogen, pulverized with a bead beater and RLT buffer was added. Total RNA was extracted using the RNeasy Micro Kit (Qiagen) with on-column DNase treatment. mRNA was purified using Dynabeads Oligo (dT).
Libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina. Final quantity was determined using the DNA TapeStation and KAPA Library Quant qPCR.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
PolyA RNA
|
| Data processing |
Raw reads were trimmed to remove low quality bases (q<10) and adapter contamination.
Gene expression levels were determined by aligning reads to the Arabidopsis thaliana genome (TAIR10) using the TopHat (v2) package and estimating read counts by the GenomicFeatures/Alignments package.
Genes responding significantly (FDR adjusted p-val <0.05) to TGA4 relative to the empty vector control were identified using DESeq2 using a ~TF+treatment model. Genes that responded to nitrogen treatment vs. the KCl control were identified for the protoplasts and whole roots using DESeq2 (FDR adjusted p-val <0.05) to compare the nitrogen response in protoplasts vs. whole roots.
Genome_build: TAIR10
Supplementary_files_format_and_content: *.csv: Read counts output from GenomicFeatures/Alignments package.
|
| |
|
| Submission date |
Mar 12, 2019 |
| Last update date |
Mar 19, 2019 |
| Contact name |
Matthew D. Brooks |
| Organization name |
USDA
|
| Department |
GCPRU
|
| Street address |
1201 W Gregory Drive
|
| City |
Urbana |
| State/province |
Illinois |
| ZIP/Postal code |
61801 |
| Country |
USA |
| |
|
| Platform ID |
GPL19580 |
| Series (1) |
| GSE128209 |
Nitrogen and cycloheximide effects on protoplast and TF-target identification in the TARGET assay |
|
| Relations |
| BioSample |
SAMN11109629 |
| SRA |
SRX5511474 |
