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Sample GSM4040041 Query DataSets for GSM4040041
Status Public on Aug 11, 2021
Title hot treated 2 hr replicate 3 [H3-2hr_S23_L003]
Sample type SRA
Source name hot treated 2 hr_leaf tissue
Organism Oryza sativa
Characteristics cultivar: IR64
treatment teperature: hot
timepoint: 2
tissue: leaf tissue
Treatment protocol To quantify transcript abundance following changes in thermal regimes, the labelled pre-existing (PE) leaves were harvested 2, 6 and 24 h after the shifting of the plants 3 h into the day period so that all sample points were collected during the day.
Growth protocol Rice (Oryza sativa) cultivar IR64 plants were grown hydroponically in a glasshouse facility at the Research School of Biology, Australian National University between October and December 2015. Seeds were initially incubated at 40-42°C for two days before soaking in water for eight hours, placed on wet Whatman filter papers in Petri dishes and kept in the dark at 30°C for five days. The germinated seedlings were then transferred to trays of vermiculite and placed in temperature-controlled glasshouses (30°C day and 25°C night) under natural sunlight and photoperiod with photosynthetically active radiation (PAR) between 400 and 1200 µmol m-2 s-1. When the third leaf had emerged, seedlings were transplanted from vermiculite to a hydroponic system. Individual plants were placed within PVC tubes with a 3.7 cm diameter and 13 cm height. Tubes were then suspended at the top of 20 L capacity hydroponic tanks (12 tanks in total), with each tank holding a maximum of 20 plants. Each tank was filled with hydroponic solution (Table S1) (Hubbart et al., 2007). The nutrient solution was replaced weekly and sulphuric acid or sodium hydroxide were used to adjust the pH to between 5-6, with pH monitored using a portable pH meter (Rowe Scientific Pty. Ltd., NSW, Australia). The hydroponic solution was aerated continuously using Infinity AP-950 air pumps (Kong’s Pty. Ltd., Ingleburn, Australia). After two weeks of hydroponic growth at 30/25°C, the most recently fully-expanded leaves were labelled as pre-existing (PE) leaves. Following labelling, four tanks were randomly chosen and shifted to an adjacent glasshouse room set to 25°C day and 20°C night (25/20°C), and four tanks to a room set at 40°C day and 35°C night (40/35°C) temperature regime, while four tanks were retained at 30/25°C as controls. Newly-developed (ND) leaves that emerged in each thermal regime were labelled, with all measurements reported on ND leaves being made 21-days after T transfer. For all experiments, four separate leaves from separate plants, one from each hydroponic tank (pot replicates) per T treatment were sampled.
Extracted molecule total RNA
Extraction protocol For each time point and temperature treatment, approximately 8 cm of middle leaf section (less than 100 mg) were sampled by immediately freezing leaf material in liquid N2 and storing at -80°C until RNA extraction. Total RNA was extracted using the RNeasy plant mini protocol (Qiagen, Doncaster, VIC, AU) and treated with DNase I (Qiagen, Doncaster, VIC, AU) to remove any contaminating DNA.
RNAseq libraries were prepared using the Illumina Stranded Total RNAseq kit with RiboZero rRNA depletion as per the manufacturer's guidelines (Illumina). Libraries were pooled and sequenced on a HiSeq1500 for 61 cycles in single end mode at the Centre for AgriBioscience, LaTrobe University, Melbourne, Australia.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 1500
Description H3-2hr_S23_L003
Data processing bcl2fastq2 Conversion Software v2.20 was used to convert bcl to fastq files and remove adapter sequences
Quality control was performed with FastQC v.0.11.2. Adapters were removed using scythe v.0.991 with flags -p 0.01 for the prior and reads were quality trimmed with sickle v.1.33 with flags -q 20 (quality threshold) -l 20 (minimum read length after trimming). The trimmed and quality filtered reads were aligned to the rice reference genome Os-Nipponbare-Reference-IRGSP-1.0 from the MSU Rice Genome Annotation Project Database v7 ( using the subjunc aligner v1.5.0-p1 with -u and -H flags to report only reads with a single unambiguous best mapping location, -P 3 for phred+33 encoding (Liao et al., 2013). Reads were then sorted, indexed and compressed using samtools v1.1-26-g29b0367 (Li et al., 2009) and strand-specific bigwig files were generated using bedtools genomecov v2.16.1 (Quinlan and Hall, 2010) and the UCSC utility bedGraphToBigWig for viewing in IGV (Robinson et al., 2011). Summary statistics for each sample are provided in Supplementary Dataset S1: Summary of transcriptomic datasets.
For standard differential gene expression testing, the number of reads mapping per IRGSP-1.0 gene loci was summarised using featureCounts v1.5.0-p1(Liao et al., 2014) with flags -P and -c to discard read pairs mapping to different chromosomes and the -s flag set to 2 for strand specificity for a strand specific library, multimapping reads and multioverlapping reads were not counted. Reads were summarised to parent IRGSP-1.0 gene loci rather than individual splice variants by summarising to the genomic coordinates defined by the feature "gene" in the IRGSP-1.0.gff reference (last modified 7/2/2012 Only loci with an abundance of at least 1 CPM (approximately 5 reads) in at least 4 samples were retained. Statistical testing for relative gene expression was performed in R following the “edgeR-limma-voom” approach (; using, edgeR v.3.4.2(McCarthy et al., 2012; Robinson and Oshlack, 2010; Robinson et al., 2010; Robinson and Smyth, 2007, 2008), and voom in the limma package 3.20.1(Law et al., 2014; Smyth, 2005, 2004).
Genome_build: Os-Nipponbare-Reference-IRGSP-1.0
Supplementary_files_format_and_content: Gene expression counts were normalised using limma-voom and are provided as a normalised counts per million per gene locus matrix.
Submission date Aug 20, 2019
Last update date Aug 11, 2021
Contact name Peter A Crisp
Organization name The University of Queensland
Department School of Agriculture and Food Science
Lab Crisp
Street address John Hines Building (62), The University of Queensland
City Brisbane
State/province QLD
ZIP/Postal code 4072
Country Australia
Platform ID GPL25149
Series (1)
GSE136045 Acclimation of rice leaves to varying temperatures
BioSample SAMN12607262
SRA SRX6743468

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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