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| Status |
Public on Feb 25, 2020 |
| Title |
IL.Drought.4 |
| Sample type |
SRA |
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| Source name |
flag-leaf tissue
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| Organism |
Oryza sativa |
| Characteristics |
tissue: Flag-leaf
developmental stage: Reproductive-stage
genotype: IL (introgression line)
phenotype: Drought-tolerant
treatment: Drought
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| Growth protocol |
A pot experiment was arranged in a randomized complete block design with two treatments (well-watered and 2-weeks drought-stressed), two genotypes, and six replications or pots in a greenhouse at the International Rice Research Institute (Los Banos, Philippines). For reproductive-stage drought stress (RDS), water was withheld at reproductive (R2) stage on discrete morphological criteria as described by Counce et al. (2000) until the soil moisture level progressively dropped to 75% field capacity (FC) and was maintained for nine days, whereas control plants were well-watered. After the 10th day, the FC was brought down and maintained at 50% for three more days. During the drought stress period, the pots were weighed daily, and the difference in weight on subsequent days was corrected by adding water to maintain the required FC. Flag-leaf samples of well-watered and drought-stressed treatments were collected at the R3 stage on the 12th day of RDS and immediately flash-frozen in liquid nitrogen. Four independent biological replicates for each tissue sample were harvested.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Flag-leaf tissue under control and drought-stress conditions were removed, flash frozen on dry liquid nitrogen, and total RNA was extracted using TRIzol reagent and isolated using the Qiagen RNeasy Plant Mini Kit (Qiagen, Limburg, Netherlands). The total RNA concentration was quantified using Nanodrop spectrophotometer (ND-1000; Nanodrop Technologies, Wilmington, DE), with absorbance at 260 nm. RNA purity and integrity were examined using an Agilent 2100 BioAnalyzer RNA 6000 Kit (Agilent Technologies). 2 ug of total RNA was submitted and Illumina library preparation and sequencing were completed following the standard protocols of Macrogen INC (Seoul, Korea).
RNA libraries were prepared for sequencing using standard Illumina protocols as per Macrogen Korea specifications.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
N_D_FL6
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| Data processing |
The Illumina Hiseq 4000 generates raw images utilizing HCS (HiSeq Control Software v3.3) for system control and base calling through an integrated primary analysis software called RTA (Real Time Analysis. v2.5.2). The BCL (base calls) binary is converted into FASTQ utilizing Illumina package bcl2fastq (v2.16.0.10). Adapters are not trimmed away from the reads.
The raw fasta reads were filtered using trimmomatic (RRID: SCR_011848; http://www.usadellab.org/cms/index.php?page=trimmomatic) with mostly default settings (ILLUMINACLIP:./TruSeq3-PE-2.fa:2:30:15 TRAILING:28 LEADING:28 MINLEN:30).
A transcriptome fasta file of the rice japonica genome fasta file and gff3 annotations downloaded from RGAP (MSUv.7) was made using the gffread function under Cufflinks (version 2.2.1) software.
Using the converted transcriptome fasta file, the Salmon software (version 0.7.2) indexed the transcriptome (Patro et al., 2017).
We then used Salmon to quantify transcript abundance in quassi-mapping mode directly using the indexed transcriptome, and the trimmed high-quality paired-end reads. The parameters -l A, -seqBias and -gcBias was provided to allow Salmon to automatically infer library type, to enable Salmon to learn and correct for sequence-specific and fragment-level GC content biases, respectively. Gene expression levels were then normalized using the transcripts per million mapped reads (TPM) method.
The estimated transcript abundance from Salmon was imported and aggregated to the gene level using the Bioconductor package tximport (version 1.2.0) (Soneson et al., 2016) complemented with the reader package (version 1.1.1) through R (version 3.3.3) software. Principal component analysis (PCA) was performed using ggplot2 (version 2.2.1) and gplots package (version 3.0.1) to determine relationships between samples.
DESeq2 software (version 1.14.1) was used in R to identify DEGs in pairwise comparisons (Love et al., 2014). Two series of differential expression (DE) analysis using the contrast argument was performed for the reference assembly approach in flag-leaf and panicle samples. Firstly, the contrasts of the condition effect for each genotype, i.e., IL_D vs IL_C, and SWA_D vs SWA_C. Secondly, the contrasts of the genotypic effect for each condition, i.e., IL_C vs SWA_C and IL_D vs SWA_D. Only genes that have at least ten reads total were used for DE analysis. The pre-filtered raw counts of the flag-leaf datasets were used for the transformation on the log2 scale, which has been normalized concerning library size using vst function in the DESeq2 package. Differentially expressed genes (DEGs) were defined as those presenting an absolute fold change (FC) ≥2 and an adjusted P-value (FDR) ≤ 0.05 in any pairwise comparison.
Genome_build: Rice Genome Annotation version 7
Supplementary_files_format_and_content: csv file in matrix table of the aggregated raw gene counts and csv files (four pairwise comparisons) with genes that passed the adjusted p-value < 0.05 correction without the log2-fold change filtering:
Supplementary_files_format_and_content: Aggregated gene counts_flag-leaf.csv: Raw gene counts of sequencing reads in comma-separated text format.
Supplementary_files_format_and_content: ILDroughtvsSwarnaDrought_gene-level_ressig-padjat0.05_vsd_filtered.csv: Gene DE analysis that passed the adjusted p-value < 0.05 correction.
Supplementary_files_format_and_content: ILControlvsSwarnaControl_gene-level_ressig-padjat0.05_vsd_filtered.csv: Gene DE analysis that passed the adjusted p-value < 0.05 correction.
Supplementary_files_format_and_content: SwarnaDroughtvsSwarnaControl_gene-level_ressig-padjat0.05_vsd_filtered.csv: Gene DE analysis that passed the adjusted p-value < 0.05 correction.
Supplementary_files_format_and_content: ILDroughtvsILControl_gene-level_ressig-padjat0.05_vsd_filtered.csv: Gene DE analysis that passed the adjusted p-value < 0.05 correction.
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| Submission date |
Feb 24, 2020 |
| Last update date |
Apr 10, 2020 |
| Contact name |
Jeshurun Asher Marayag Tarun |
| E-mail(s) |
J.Tarun@irri.org
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| Phone |
+632858056002288
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| Organization name |
International Rice Research Institute
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| Department |
Plant Breeding
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| Lab |
Genetics and Genomics Lab
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| Street address |
Pili Drive
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| City |
Los Banos |
| State/province |
Laguna |
| ZIP/Postal code |
4031 |
| Country |
Philippines |
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| Platform ID |
GPL23013 |
| Series (2) |
| GSE145868 |
Comparative transcriptomics and co-expression networks reveal tissue- and genotype-specific responses to reproductive-stage drought stress in rice (Oryza sativa L.) [flag-leaf] |
| GSE145870 |
Comparative transcriptomics and co-expression networks reveal tissue- and genotype-specific responses to reproductive-stage drought stress in rice (Oryza sativa L.) |
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| Relations |
| BioSample |
SAMN14177868 |
| SRA |
SRX7797216 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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