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| Status |
Public on Aug 20, 2014 |
| Title |
wheat salinity stressed (WSS) |
| Sample type |
SRA |
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| Source name |
7-day old whole seedling
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| Organism |
Triticum aestivum |
| Characteristics |
cultivar: PBW343
tissue: 7-day old whole seedling
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| Treatment protocol |
For the preparation of tissue-specific small RNA libraries, shoot and root tissues were collected separately from hydroponically grown seven-day-old seedlings. Mature leaf and spikelets were collected from field-grown plants 50 days after planting (DAP) and 90 DAP, respectively. Seven-day-old seedlings were employed for abiotic stress-related studies. For heat stress, seedlings were subjected to 35ºC and 40ºC for 30 min, 2 h, 4 h and 8 h each. Salinity stress was induced by treating the seedlings with 150 mM and 250 mM saline solution (sodium chloride) for 3 h, 6 h, 12 h and 24 h each. For water-deficit stress, seedlings were exposed to 20% PEG6000 (polyethylene glycol) and 400 mM mannitol for 1 h, 3 h, 6 h and 12 h each. Tissues for all time-points of each stress were pooled to obtain four samples, which are referred to as: HS (heat stress), SS (salinity stress), WDS (water-deficit stress). A control sample (C) was kept as wheat seedlings grown under controlled conditions along with all the abiotic stress treatments.
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| Growth protocol |
Wheat (Triticum aestivum cv. PBW343) seeds were surface-sterilized with 4% (v/v) sodium hypochlorite solution followed by 5-6 washes with sterile water and planted on muslin cloth for growing hydroponically in controlled conditions (temperature: 25±1ºC; relative humidity: 70-75%; photoperiod: 16h light/8h dark) in a growth room.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from various tissues following a modified protocol by Chomzynski and Sacchi (1987). Lithium chloride method was employed for the enrichment of low molecular weight RNA (LMW) fraction (Katiyar-Agarwal and Jin, 2007). Concentration of RNA was determined using spectrophotometer (Bio-Rad, USA) followed by evaluating the quality on MOPS-formaldehyde-agarose gel (total RNA) or TBE-urea-PAGE (LMW RNA).
For small RNA library construction, 50 µg of LMW RNA was resolved on 15% denaturing polyacrylamide gel and sRNAs in the size-range of 18-40 nt were purified from the gel followed by sequential ligation with 5’ adapter and 3’ adapter. After each adapter ligation, size selection was performed using a polyacrylamide gel and ligated product was eluted from the gel. Subsequently, first strand cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen, USA) and 3’ adapter-specific RT primer. The cDNA was amplified using adapter-specific sequencing primers and the amplified product was purified. Prior to sequencing, quality and quantity of the amplified small RNA-cDNA libraries was evaluated on Agilent 2100 Bioanalyzer system (Agilent Technologies, USA). Sequencing was performed using Illumina GAIIx sequencing platform at High-throughput Sequencing Facility, University of Delhi South Campus, New Delhi, India according to manufacturer’s instructions (Illumina, USA). All the adapters, RT primer and sequencing primers were procured from Illumina, USA.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
Illumina Casava1.7 software used for basecalling.
Purity filtered raw reads were analyzed using Java-based UEA Small RNA Workbench version 2.4 (Stocks et al. 2012). Due to the unavailability of whole genome sequence of Triticum aestivum, sequence datasets from several resources (BACs, GSSs, ESTs available at NCBI and 5X coverage wheat genomic dataset available as EMBL/Genbank SRA accession number ERP000319) were compiled as ‘in-house wheat genome dataset’ to map putative sRNAs. UEA sRNA workbench v2.4 was employed for prediction of miRNAs and miRBase v20 database was used as a reference for identification of conserved, variants of conserved miRNAs and novel miRNAs.
Genome_build: BACs, GSSs, ESTs available at NCBI and 5X coverage wheat genomic dataset available as EMBL/Genbank SRA accession number ERP000319
Supplementary_files_format_and_content: Text files with abundance measurements
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| Submission date |
Dec 19, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Gopal Joshi |
| E-mail(s) |
gopal_joshii@yahoo.com
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| Phone |
+91-11-27662609
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| Organization name |
Delhi University
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| Department |
Department of Botany
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| Lab |
Plant Genomics & Stress Biology
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| Street address |
First Floor
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| City |
New Delhi |
| State/province |
Delhi |
| ZIP/Postal code |
110007 |
| Country |
India |
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| Platform ID |
GPL15632 |
| Series (1) |
| GSE53487 |
A Comprehensive Genome-wide Study on Tissue- and Abiotic Stress-specific miRNAs in Triticum aestivum |
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| Relations |
| BioSample |
SAMN02469076 |
| SRA |
SRX396208 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1294658_Processed_WSS.txt.gz |
19.4 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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