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Sample GSM4040028

Query DataSets for GSM4040028

Status

Public on Aug 11, 2021

Title

cold treated 2 hr replicate 2 [C2-2hr_S32_L004]

Sample type

SRA

Source name

cold treated 2 hr_leaf tissue

Organism

Oryza sativa

Characteristics

cultivar: IR64
treatment teperature: cold
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

C2-2hr_S32_L004

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 (http://rice.plantbiology.msu.edu/) 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 ftp://ftp.plantbiology.msu.edu/pub/data/Eukaryotic_Projects/o_sativa/annotation_dbs/pseudomolecules/version_7.0/all.dir/all.gff3). 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 (https://www.bioconductor.org/help/workflows/RNAseq123/); 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

E-mail(s)

p.crisp@uq.edu.au

Organization name

The University of Queensland

Department

School of Agriculture and Food Science