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Sample GSM1716999

Query DataSets for GSM1716999

Status

Public on Dec 31, 2016

Title

Lalat_grain [AFG2]

Sample type

SRA

Source name

Central Rice Research Institute Gene Bank

Organism

Oryza sativa

Characteristics

cultivar: Lalat
grain iron conc.: lower concentration (7.80 ppm) of Fe in the grain
crop duration: 100-110 days
tissue: Long slender rice grain

Growth protocol

Seeds were surface sterilized by immersing them in a 4% solution of commercial sodium hypochlorite solution for 10 min, followed by repeated washing with glass-distilled water. Treated seeds were allowed to germinate at 30oC in darkness in a petri dish lined with two layers of moistened filter paper discs. One week old seedlings were transferred to plastic pots filled soil and farm yard manure in 3:1 ratio and grown in the net house under ambient conditions till maturity. Fifteen days after panicle emergence (mid-grain-filling stage) roots and developing grains were collected, washed in sterile nanopure water, blotted, dipped in RNA stabilizer solution in a falcon tube and stored in refrigerator for 24h. The tubes were sifted to -80oC ultra freezer for until future use.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from roots and developing rice grains (sampled 15 days after panicle emergence) using Plant RNA miniprep kit (Xcerlis Genomis, India) as per standard protocol provided with the kit. The yield and purity of RNA were assessed by determination of the absorbance at 260 and 280 nm. Further, RNA quality was checked using RNA 6000 Nano Assay Kit and Agilent Bioanalyzer 2100. RNA Integrity Number (RIN) values were >8.5 for all samples. The pair end Sequencing libraries were prepared using Illumina TruSeq® RNA Library Preparation Kit as per manufacturer's protocol (Illumina®, San Diego, CA).
Total RNA (4µg) from each sample was used to isolate poly(A) mRNA. Steps involved in library preparation were mRNA fragmentation, first strand cDNA synthesis followed by second strand cDNA synthesis and adaptor ligation, 200-bp cDNA fragments isolation and amplification by 18 cycles of PCR. Library quality control and quantification were performed on Caliper LabChip GX using HT DNA High Sensitivity Assay Kit. The libraries of all samples were of 285 bp to 303 bp range.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

grain transcriptom of Lalat cultivar

Data processing

Cluster generation and sequencing: RNA-Seq was performed using Illumina HiSeq2000 instrument. Amplified products were loaded on sample loading port of eight tube strip on cBOT for cluster generation and further subjected to 100 cycles of paired-end (2 × 100 bp) sequencing on HiSeq 2000. Approximately, 30 million reads were generated per sample. The processing of fluorescent images into sequences, base-calling and quality value calculations were performed using the Illumina data processing pipeline (version 1.8).
Read mapping: Rice genome and gene information for reference cultivar Nipponbare (Oryza sativa L. subsp. japonica) was downloaded from ftp://ftp.plantbiology.msu.edu/pub/data/Eukaryotic_Projects/o_sativa/annotation_dbs/pseudomolecules/version_7.0/all.dir/ Further, raw reads were filtered to obtain high-quality reads by removing low-quality reads with Q < 25. After trimming low-quality reads and adaptor sequences, the resulting high-quality reads were mapped onto the downloaded reference genome. Sequencing reads from all 4 transcriptome libraries were first pooled and mapped to the rice genome sequence scaffolds using the Bowtie (V 0.12.9) and TopHat (V 1.3.3) with default parameters. The resulting alignment (in BAM file format) was used to generate transcript annotations (in gene feature file or GFF format) with the Cufflinks (V 1.3.0). The Ensembl reference annotation in GFF format was used to provide established junctions via the ‘–G’ option. These read counts were used in statistical tests of differential expression between test and reference for differential gene expression [PMID:22383036; PMID: 22853646].
Assessment of differentially expressed genes (DEGs): Differences in gene expression between different samples were tested with Cuffdiff package of Cufflinks using FPKM (Fragments Per Kilobase of transcript per Million mapped reads) from reference-guided mapping. Genes expressed at very low levels (read counts < 10 across all six libraries) were not used in analysis of differential gene expression. Significance tests for differential expression were based on a modified exact test. A false discovery rate (FDR) of 0.05 and p-value ≤0.05 was used for identifying differentially expressed genes. The CuumeRbund, a package of R was used to generate the heatmaps of differentially expressed genes [PMID:22383036; PMID: 22853646].
Genome_build: Rice genome and gene information for reference cultivar Nipponbare (Oryza sativa L. subsp. japonica) was downloaded from ftp://ftp.plantbiology.msu.edu/pub/data/Eukaryotic_Projects/o_sativa/annotation_dbs/pseudomolecules/version_7.0/all.dir/

Supplementary_files_format_and_content: MS Excel, Supplementary file 1: Number of genes that are differentially expressed in the roots and developing grains of the cultivars Sharbati and Lalat. Gene with FPKM values obtained by Cufflink v.1.3.0 higher than zero was considered as expressed genes.
Supplementary file 2: List of Gene ontology terms that are enriched for genes expressed in four samples. P-value less than 0.05 were considered as significant.
Supplementary file 3: List of differential expressed genes enriched in pathway analysis performed by KAAS online pathway annotation.
Supplementary file 4: List of transcription factors analysed by BlastX search against rice transcription factor database
Supplementary file 5: Complete list of rice novel transcripts that are expressed and their putative functions in AFR1 in the roots and developing grains of the cultivars Sharbati and Lalat.

Submission date

Jun 22, 2015

Last update date

May 15, 2019

Contact name

Avijit Das

E-mail(s)

avijitcrri@gmail.com

Phone

09432370939

Organization name

NIRJAFT