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Sample GSM954259 Query DataSets for GSM954259
Status Public on Jul 13, 2017
Title wild-type under drought stress (dsur)
Sample type SRA
Source name wild-type
Organism Arabidopsis thaliana
Characteristics application: 5' end-seq of uncapped RNA
stess: 2 hr drought
genotype/variation: wild-type
Treatment protocol Two-week-old plants were subjected to drought stress treatment as described previously (Matsui et al. 2008, Plant Cell Physiol, 49: 1135-1149).
Growth protocol Arabidopsis rdr1/2/6 and wild-type plants were grown on MS medium at 22 ºC in the cycle of 16hr light / 8hr dark condition for 2 weeks as described previously (Matsui et al. 2008, Plant Cell Physiol, 49: 1135-1149).
Extracted molecule total RNA
Extraction protocol rRNAs were removed from 100 μg of total RNA twice using RiboMinus Plant Kit for RNA-Seq (Life Technologies). The rRNA-depleted RNAs were ligated with 200 pmol of 5'-end RNA (adaptor 1) by T4 RNA ligase (Takara) at 37 °C for 30 min. 5’ RNA adaptor includes EcoP15I site and P2 site for SOLiD sequencing. Only uncapped RNAs can be ligated to 5’ RNA adaptor at this step. The adaptor-ligated RNAs were subjected to synthesis of single-stranded cDNAs using 100 pmol of RT primer (primer 1) that containing EcoP15I site and random hexamer by SuperScript III (Life Technologies). The single-stranded cDNAs were amplified to double-stranded cDNAs by PCR (94 ˚C, 3 min; (94 ˚C 30 sec; 55 ˚C, 30 sec; 72 ˚C, 8 min) ×10 cycles), using biotin-labled 1st PCR forward primer (primer 2) and 1st PCR reverse primer (primer 3). The PCR products were digested by EcoP15I (NEB) at 37 °C for 16 hr.  The 5’-end fragments were recovered by biotin capture beads (Takara) and ligated with 3’-end dsDNA adaptor (adaptor 2) at 16 °C for 2 hr. The 3’ adaptor-ligated cDNAs were purified and amplified by PCR {94 ˚C, 3 min; (94 ˚C, 30 sec; 55 ˚C, 30 sec; 72 ˚C, 30 min) ×10 cycles} using 2nd PCR forward primer and reverse primer (primers 4 and 5). Finally, PCR products (120-130 bp) were recovered and subjected to SOLiD sequencing. Adaptors: 1. 5'-end RNA adaptor: 5'-CTGCTGTACGGCCAAGGCGCAGCAGGG-3', 2. 3’-end dsDNA adaptor 5'-NNTAGTGGCTGACGGGTATCTCTCCTTTCG CCTCCGCATCACC-3', Primers: 1. RT primer: 5’-GTCTCTAGCCTGCAGGATCGATCTATCAGCAGNNNNNNNN-3', 2. Biotin-labled 1st PCR forward primer: 5’-Biotin-CTGCTGTACGGCCAAGGCGCAGCA-3', 3. 1st PCR reverse primer: 5'-GTCTCTAGCCTGCAGGATCGAT-3', 4. 2nd PCR forward primer: 5'-CTGCCCCGGGTTCCTCATTCTCT******CTGCTGTACGGCCAAGGCGCA-3 (******* : barcode sequence), 5. 2nd PCR reverse primer:5'-CCACTACGCCTCCGCTTTCC-3'
Library strategy RNA-Seq
Library source transcriptomic
Library selection size fractionation
Instrument model AB SOLiD 4 System
Data processing 17nt RNA sequences obtained from SOLiD sequencing were mapped on Arabidopsis reference genome. If sequence tags are mapped on more than 6 loci, then the sequences are removed. Mapped tag no. of each sample was adjusted to 50 million.
Genome_build: TAIR10 Arabidopsis genome annotation was used.
Supplementary_files_format_and_content: Tag no. was displayed as variablestep in wiggle (WIG) format file. The position of AGI-annotated genes was shown in BED file.
Submission date Jun 29, 2012
Last update date May 15, 2019
Contact name Motoaki Seki
Organization name RIKEN CSRS
Street address 1-7-22, Suehiro-cho, Tsurumi
City Yokohama
ZIP/Postal code 230-0045
Country Japan
Platform ID GPL14599
Series (2)
GSE39033 Deep sequencing of uncapped RNA in rdr1/2/6 and wild-type Arabidopsis plants
GSE39037 Deep sequencing of small and uncapped RNA in rdr1/2/6 and wild-type Arabidopsis plants
SRA SRX157450
BioSample SAMN01085318

Supplementary file Size Download File type/resource
GSM954259_DSUR_WT_Dry_Minus.wig.gz 20.3 Mb (ftp)(http) WIG
GSM954259_DSUR_WT_Dry_Plus.wig.gz 20.3 Mb (ftp)(http) WIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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