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| Status |
Public on Jul 13, 2017 |
| Title |
rdr1/2/6 under drought stress (dsur) |
| Sample type |
SRA |
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| Source name |
rdr1/2/6
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| Organism |
Arabidopsis thaliana |
| Characteristics |
application: 5' end-seq of uncapped RNA
stress: 2 hr drought
genotype/variation: rdr1/2/6
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| 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).
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| 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).
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| 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'
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
AB SOLiD 4 System |
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| 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.
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| Submission date |
Jun 29, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Motoaki Seki |
| E-mail(s) |
motoaki.seki@riken.jp
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| Organization name |
RIKEN CSRS
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| Street address |
1-7-22, Suehiro-cho, Tsurumi
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| City |
Yokohama |
| ZIP/Postal code |
230-0045 |
| Country |
Japan |
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| 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 |
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| Relations |
| SRA |
SRX157452 |
| BioSample |
SAMN01085320 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM954261_DSUR_rdr126_Dry_Minus.wig.gz |
15.2 Mb |
(ftp)(http) |
WIG |
| GSM954261_DSUR_rdr126_Dry_Plus.wig.gz |
15.2 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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