 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Nov 09, 2012 |
| Title |
11dpi infected |
| Sample type |
SRA |
| |
|
| Source name |
virus-infected tobacco leaves
|
| Organism |
Nicotiana tabacum |
| Characteristics |
plant variety: cv. Xanthi nc
time: 11dpi
tissue: leaves
viral infection: Cucumber mosaic virus M strain
|
| Treatment protocol |
The systemically infected leaves of M-CMV infected plants were harvested and immediately homogenized to prepare viral inoculum. Approximately 0.5 g leaves were homogenized with kieselguhr in 1 ml water. The viral inoculum was rub-inoculated by a latex-gloved finger onto the top two leaves of tobacco plants having four to six fully expanded leaves. The mock inoculum was prepared from leaves of healthy plants and applied in the same way as viral inoculum.
|
| Growth protocol |
Tobacco plants (Nicotiana tabacum cv. Xanthi nc) were grown in a greenhouse at a cycle of 16 h light and 8 h dark.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The systemically infected leaves and mock-inoculated leaves were harvested at 6, 9, 11, 13, 16 and 20 days post-inoculation (dpi), respectively. In order to average out variation of different plants, five leaves from five different plants were mixed to prepare every RNA sample. The collected leaf tissues were frozen in liquid nitrogen and stored at -80â until RNA extraction. Total RNAs of leaf tissues were extracted using TRIzol® Reagent and then treated with DNase I. For DGE profiling, twelve individual tag libraries of samples (six infected samples and six mock-inoculated samples) were constructed in parallel. Sequence tags were prepared using Illumina Gene Expression Sample Prep Kit following the manufacturer's instructions. Magnetic beads with oligo(dT) were used to purify poly(A)-containing mRNA and oligo-dT primer was used to sythesize double-stranded cDNA. The bead-bound cDNAs were then digested by endonuclease NlaIII, which recognizes and cuts CATG sites, to generate sticky 5' ends. The cut-off fragments were washed away and the bead-bound fragments were ligated to the Illumina adaptor 1 through the sticky 5' end. The bead-bound fragments with adaptor 1 were then digested by MmeI, which cuts at 17bp downstream of CATG site. After that, the bead-bound fragments without adaptor 1 were removed by magnetic beads precipitation, and the tags with adaptor 1 were purified and ligated to Illumina adaptor 2 through the 3' end of the tag. After 15 cycles of linear PCR amplification, 95bp fragments were purified by 6% TBE polyacrylamide gel electrophoresis to obtain the final tag libraries. After denaturation, the single-stranded DNAs were fixed onto the Illumina Sequencing Chip (flowcell). Four types of nucleotides labeled by four colors were used and sequencing was performed with the method of sequencing by synthesis (SBS) using Illumina HiSeq⢠2000.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
*_TagCount.txt: Extraction of tags and tag counting were performed using the Illumina pipeline. To get the high quality tags that we used to analysis, we filtered the potentially erroneous tags (only one copy number, only adaptor sequences, or containing unknown sequences 'N') to get the clean tags. For mapping the clean tags to reference sequences, we get all the possible 17 bases length sequences next to the NlaIII restriction site of the reference sequences, then plus the 4 bases NlaIII recognition site to construct a reference database. The clean tag expression was normalized to TPM (transcripts per million clean tags).
Unigene.blast.Nr.xls, Unigene.blast.Swissprot.xls, Unigene.blast.kegg.xls, Unigene.blast.cog.xls: The Unigenes were analyzed by searching the protein databases like nr, Swiss-Prot, KEGG and COG using blastx (evalue < 0.00001) program. If results of different databases conflicted with each other, a priority order of nr, Swiss-Prot, KEGG and COG should be followed when deciding sequence direction of Unigenes.
Unigene.gene2GO.xls, Unigene.GO2gene.xls: Unigenes with nr annotation were further analyzed with Blast2go to get GO annotations.
|
| |
|
| Submission date |
Jun 22, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Jie Lu |
| E-mail(s) |
lujiebio@gmail.com
|
| Organization name |
CAIQ
|
| Street address |
Shuangqiaozhonglu
|
| City |
Beijing |
| ZIP/Postal code |
100121 |
| Country |
China |
| |
|
| Platform ID |
GPL13760 |
| Series (1) |
| GSE30162 |
Digital gene expression (DGE) of CMV-infected tobacco leaves |
|
| Relations |
| SRA |
SRX080204 |
| BioSample |
SAMN00631243 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM746924_3b_TagCount.txt.gz |
1.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
