|
| Status |
Public on Sep 04, 2014 |
| Title |
Col20GC2 |
| Sample type |
SRA |
| |
|
| Source name |
Col green cotyledon stage seeds
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Col
Stage: Green cotyledon stage developing seed
maturation temperature: 20°C
time point: 5-7hrs after dawn
|
| Growth protocol |
Plants were grown until flowering at 22°C under standard long days using fluorescent white light at 80 to 100 μmolm−2s−1 until anthesis of the first flowers. Once flowering, plants were transferred to growth cabinets running the same conditions, but with the indicated seed maturation temperatures and left until harvesting at CT5 - CT7
|
| Extracted molecule |
total RNA |
| Extraction protocol |
150 mg of seed (based on dry seed weight) was ground and extracted with 1 ml of frozen XT buffer (0.2M sodium borate, 30 mM EGTA, 1% SDS, 1% sodium deoxycholate, 2% polyvinylpyrollidone, 10 mM DTT, and 1% IGEPAL [pH 9.0]) in a pestle and mortar. This was allowed to thaw and was treated with 40 μl proteinase K (PCR grade, Roche, UK) for 90 min at 42°C; precipitation on ice followed for 1 hr with 80 μl 2M potassium chloride. The supernatant was collected after centrifugation at 4°C. The RNA was precipitated from the supernatant at −20°C for 2 hr with 360 μl 8M lithium chloride. The RNA was collected by centrifugation at 4°C and redissolved in 100 μl water. The RNA was further purified via the clean-up protocol of the RNeasy Plant RNA isolation kit (Qiagen) according to the manufacturer's protocol.
5 μg RNA was submitted to sequencing using standard protocols for Illumina TruSeq™ technologies RS-122-2001 | TruSeq™ RNA Sample Prep Kit v2. Data were generated on Illumina HiSeq 2000. The input material contains 1-2% PhiX which is used as a control.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Samples are pre-filtered using Bowtie to remove reads matching PhiX control sequence.
Samples were then filtered using the fastq-mcf program from the ea-utils package (http://code.google.com/p/ea-utils/)
Spike-in reads were removed using Bowtie and the lower limit of detection quantified using custom scripts.
The remaining reads were aligned using Tophat v1.4.1 and the following parameters -I 10000 -r 50 --mate-std-dev 100 –library-type fr-unstranded.
The Cufflinks package was used to quantify gene and isoform abundance using the -G flag. The 17 cuffcompare and cuffdiff components of Cufflinks were used to quantify gene and isoform differential expression (Trapnell et al., 2012).
All other settings were as per the default settings. Further analysis was performed using the cummeRbund package (Trapnell et al., 2012).
Genome_build: TAIR9: GCF_000001735.2
Supplementary_files_format_and_content: TopHat output files accepted_hits.bam and CuffLinks output files _genes.fpkm_tracking
|
| |
|
| Submission date |
Sep 03, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Dana Reid MacGregor |
| E-mail(s) |
dana.macgregor@rothamsted.ac.uk
|
| Organization name |
Rothamsted Research
|
| Department |
Biointeractions and Crop Protection
|
| Street address |
Rothamsted Research
|
| City |
Harpenden |
| State/province |
Hertfordshire |
| ZIP/Postal code |
AL5 2JQ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL13222 |
| Series (1) |
| GSE61061 |
Dormancy of green-cotyledon Col seeds matured at 20°C vs 15°C |
|
| Relations |
| BioSample |
SAMN03018420 |
| SRA |
SRX691585 |
