|
| Status |
Public on Jun 20, 2014 |
| Title |
Mutant |
| Sample type |
SRA |
| |
|
| Source name |
Five-day-old seedlings, wbc19 mutant, no kanamycin
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
line: SALK_107731
genotype/variation: wbc19 mutant
age: 5 days
tissue: seedling
growth media: MS
|
| Treatment protocol |
Homozygous seeds were plated under sterile conditions on MS media with or without kanamycin 50mg/l.
|
| Growth protocol |
Control (SALK_064816C) and wbc19 mutant (SALK_107731) lines were used. SALK_064816C mutants served as controls as they carry a T-DNA insertion that does not disrupt a gene. The T-DNA insertion is located in the vicinity of the TRANSPARENT TESTA 4 gene, 260 bp upstream of the start codon and the mutants have no visible phenotype. After stratification (3 days at 4°C), plates were transferred to a controlled-environment cabinet (23°C, 16 h light; 18°C, 8 h dark) and plant material was harvested and flash frozen 5 days later.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using a Plant RNeasy Mini Kit (Qiagen) and poly(A) mRNA isolated using oligo(dT) DynaBeads (Life Technologies).
Messenger RNA fragmentation and RNA-seq library generation were according to Guo et al., 2010 (Nature 466: 835-840). Briefly, after alkaline fragmentation of mRNA, ~25-45 mer fragments were gel purified on a 10% denaturing polyacrylamide-urea gel. After 3'-dephosphorylation with polynucleotide kinase (PNK, New England Biolabs), a 3' adaptor was ligated to the fragments. Following a second gel purification step, the fragments were 5' phosphorylated with PNK and the 5' adapter ligated. Each library was then amplified for 21 cycles and gel purified on a 90% formamide, 8% acrylamide denaturing gel.
RNA-seq: single-end sequencing on the Illumina GAII high-throughput sequencing platform.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
mRNA
|
| Data processing |
Processed GSE58662_READs using FASTX clipper version 0.0.6 to remove adapter sequences and keep sequences of 36 mer.
Processed GSE58662_READs were aligned to the Arabidopsis thaliana reference genome (TAIR10, ENSEMBL 14) using TopHat version 2.0.5. The minimum GSE58662_READ length was set to 18 and up to 2 mismatches were tolerated.
Mapped GSE58662_READs were assembled and merged using Cufflinks version 2.0.2 and Cuffmerge 2.0.2.
Transcript abundance and differential expression was analyzed using Cuffdiff version 1.3.0. Statistical significance was only tested when a minimum of 10 GSE58662_READs were mapped on a given transcript.
Genome_build: TAIR10
Supplementary_files_format_and_content: .xls file with RPKM values for each sample and p value generated by Cuffdiff; gene descriptions and GO classifications were obtained from The Arabidopsis Information Resource (TAIR). Section and column definitions are in the GSE58662_README.txt file.
|
| |
|
| Submission date |
Jun 19, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Mentewab Ayalew |
| E-mail(s) |
mayalew@spelman.edu
|
| Phone |
404 270 5712
|
| Organization name |
Spelman College
|
| Department |
Biology Department
|
| Street address |
350 Spelman Lane
|
| City |
Atlanta |
| State/province |
GA |
| ZIP/Postal code |
30314 |
| Country |
USA |
| |
|
| Platform ID |
GPL9302 |
| Series (1) |
| GSE58662 |
RNA-seq analysis of the effect of kanamycin and the ABC transporter AtWBC19 on Arabidopsis thaliana seedlings reveals changes in metal content |
|
| Relations |
| BioSample |
SAMN02867534 |
| SRA |
SRX611342 |
