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Sample GSM5128897

Query DataSets for GSM5128897

Status

Public on Apr 21, 2021

Title

AuWH_C

Sample type

SRA

Source name

Seedling shoot tissue

Organism

Triticum turgidum subsp. durum

Characteristics

genotype: DBA Aurora
parental treatment: Water-deficit & heat stress group parents
progeny treatment: Control group

Treatment protocol

Complete basal nutrient solution was provided to progeny control treatment groups (AuCG_C, AuWH_C, L6CG_C, L6WH_C). The progeny N starvation treatment groups (AuCG_N, AuWH_N, L6CG_N, L6WH_N) were supplied with the basal nutrient solution without the nitrogen component.

Growth protocol

Young seedlings were grown in controlled glasshouse environment (24/18 °C, 16/8 h photoperiod). Each treatment group had 12 individual biological replicates (one plant per pot). Each pot contained 1.2 kg of N40 sand with 0.5% CaCO3.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from frozen seedling shoot material using the Tri reagent as previously described (Sigma-Aldrich, North Ryde, Australia). Extracted total RNA samples were treated with TURBO DNase (ThermoFisher Scientific, Scoresby, Australia) following the manufacturer’s instructions. The concentration, quantity and quality of purified total RNA samples were measured on a NanoDrop Lite spectropho-tometer (ThermoFisher Scientific, Scoresby, Australia). RNA integrity was assessed by agarose gel electrophoresis and Bioanalyzer measurement. High-quality RNA samples from the six biological replicates per treatment group were equally pooled for small RNA sequencing purpose.
High-quality RNA samples from the six biological replicates per treatment group were equally pooled for small RNA sequencing purpose. A total of eight small RNA libraries (AuCG_C, AuCG_N, AuWH_C, AuWH_N, L6CG_C, L6CG_N, L6WH_C, L6WH_N) were made using the TruSeq Small RNA Sample Prep Kit
Small RNA sequencing (single-end, 50 bp) was performed on an Illumina NovaSeq 6000 at LC-Bio (Hangzhou, China).

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina NovaSeq 6000

Data processing

Small RNA sequencing analysis was performed using the ACGT101-miR program (LC Sciences, TX, USA). Raw reads were first processed by removing low-quality reads and adapter sequences to obtain clean sequences. Reads with a nucleotide (nt) length <18 nt or >25 nt were removed.
Non-coding (nc) RNA families (rRNA, tRNA, snRNA and snoRNA), repeats and mRNA sequences were discarded using RFam, Repbase and durum wheat NCBI mRNA entries as references. Unique sRNA sequences were then obtained for each library.
Conserved mature miRNAs and 5p- or 3p-derived miRNA variants were identified using BLAST search against the plant miRBase (Release 22.1). MIR (microRNA gene locus) and miRNA sequences from common plant species in the miRBase were used as references. Sequences mapped to the mature miRNA in the hairpin were identified as conserved mature miRNAs. Sequences mapped to the opposite arm of mature miRNA in the hairpin were identified as 5p- or 3p-derived variants. Single mismatch within the sequence and length variation at both 5p and 3p were allowed in the alignment.
All the mapped miRNAs were aligned to the durum wheat genome (NCBI UID 3439611, assembly Svevo.v1) to determine their genomic location. The remaining unmatched sRNA sequences were used to identify novel durum miRNAs. Sequences were BLASTed to the durum wheat genome.
To identify miRNA precursors, secondary hairpin structures containing matched sequences were predicted using RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) from 120nt of flanking genome sequences.
Genome_build: Durum wheat genome (NCBI UID 3439611, assembly Svevo.v1)
Supplementary_files_format_and_content: Matrix table with normalized reads count of each microRNA in each library

Submission date

Mar 02, 2021

Last update date

Apr 21, 2021

Contact name

Haipei Liu

E-mail(s)

haipei.liu@adelaide.edu.au

Organization name

The University of Adelaide

Street address

PMB1 Waite Building, School AFW