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| Status |
Public on Nov 26, 2013 |
| Title |
Na |
| Sample type |
SRA |
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| Source name |
NaHCO3-treated cucumber leaves
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| Organism |
Cucumis sativus |
| Characteristics |
strain: Jinchun 5
tissue: leaves
time: 12d after treatment
treatment: NaHCO3
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| Growth protocol |
Cucumber plants were grown in a greenhouse at a cycle of 16 h light and 8 h dark.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Samples from Na- and SNP-treated leaves were harvested and immediately frozen in liquid nitrogen, and stored at -80°C. Total RNA was isolated from the frozen leaf with an RNA simple Total RNA Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. For DGE profiling, the two individual tag libraries of samples (Na sample and SNP sample) 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 HiSeqTM 2000.
RNA libraries were prepared for sequencing using standard Illumina protocols
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Illumina RTA 1.13.48 software used for basecalling.
*_TagCopyNumber.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).
Clean tags were mapped to cucumber genome V1 and the tags mapped to one gene were designed as unambiguous clean tags. The number of unambiguous clean tags for each gene was calculated and then normalized to TPM to better measure gene expression level. The genes were analyzed by searching the protein databases nr using blastx (evalue < 0.00001) program. and further analyzed with Blast2go to get GO annotations.
Genome_build: Cucumber genome V1 (http://www.ncbi.nlm.nih.gov/assembly/GCA_000004075.1/)
Supplementary_files_format_and_content: xls files containing gene accession gene expression level, GO analysis, blast nr, mapped-tag and its number.
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| Submission date |
Nov 21, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Zhongxi Gao |
| E-mail(s) |
[email protected]
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| Phone |
86-0538-8241447
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| Organization name |
Shandong Agricultural University
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| Street address |
Daizong street no. 61
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| City |
Tai'an |
| ZIP/Postal code |
271018 |
| Country |
China |
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| Platform ID |
GPL16310 |
| Series (1) |
| GSE42439 |
Digital gene expression (DGE) of alkali-stressed cucumber leaves |
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| Relations |
| SRA |
SRX206337 |
| BioSample |
SAMN01818726 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1040131_Na_GeneExpression.txt.gz |
471.2 Kb |
(ftp)(http) |
TXT |
| GSM1040131_Na_TagCopyNumber.txt.gz |
585.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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