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Sample GSM2184186

Query DataSets for GSM2184186

Status

Public on Jun 01, 2017

Title

J2_RNA-seq

Sample type

SRA

Source name

Callus

Organism

Oryza sativa Japonica Group

Characteristics

rice cultivar: Tainung 67
tissue culture stage: Subculture
time point: T2

Treatment protocol

The callus samples were collected at different time points of culture starting with embryonic stage (T0). N6D medium was used for callus induction of both cultivars; induced calli were transferred to fresh N6D medium for subsequent callus growth. Finally calli were transferred to REIII M2 medium (MS salt with vitamin, 1g/L casein hydrolysate, 3% sucrose, 3% sorbitol, 0.02 mg/L NAA, 2mg/L BAP, pH5.8 0.4% gelrite) for regeneration.

Growth protocol

Seeds of TNG67 and IR64 were surface-sterilized in 70% ethanol for 1 min and rinsed with distilled water. The sterilized seeds were soaked in 2.5% sodium hypochlorite (NaClO; The Clorox Co., Oakland, CA, USA) containing 0.2% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA) for 15 min in a rotator followed by washing with sterile ddH2O. After surface sterilization, seeds were sown on N6D media (N6 salt, Vitamin B5, 3% sucrose, 2.5 mg/L 2,4-D pH 5.8 0.4% gelsite) for callus induction.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted & quantified using Qubit RNA assay 100 ng RNA was used as starting material for each sample. Total RNA are treated with DNase I (Roche), and cleaned up with phenol-chloroform and precipitated with ethanol.
RNA–Seq libraries were prepared following standard Illumina protocols. Resulted libraries were then subjected to PCR amplification prior to sequencing on the Illumina HiSeq 2000 sequencers (at National Center for Genome Medicine, Academia Sinica). The quality of reads was checked with FastQC (Andrews, 2010), reads were mapped to Os-Nipponbare-Reference-IRGSP-1.0 using Bowtie (Langmead et al., 2009) allowing two mismatches and only unique alignments were retained. The resulted alignment file was processed and differential expression was calculated using Tophat (Langmead et al., 2009) to generate Fragments Per Kilo Base Per Million mapped reads (FPKM) per gene by applying 4-fold cut-off.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

RNA-Seq

Data processing

The quality of reads was checked with FastQC (Andrews, 2010), reads were mapped to Os-Nipponbare-Reference-IRGSP-1.0 using Bowtie (Langmead et al., 2009) allowing up to two mismatches and only unique alignments were retained. The alignment file was processed and differential expression was calculated using Tophat (Langmead et al., 2009) to generate Fragments Per Kilo Base Per Million mapped reads (FPKM) per gene by applying 4-fold cut-off .
We identified differentially expressed genes (DEGs) using Tophat to generate Fragments Per Kilo Base Per Million mapped reads (FPKM) per gene. The transcripts showing at least four-fold change with P-value less than 0.05 were identified as DEGs.
genome build: MSU7
processed data files format and content: Excel file with FPKM values.

Submission date

Jun 01, 2016

Last update date

May 15, 2019

Contact name

Moloya Gohain

E-mail(s)

moloyandri@gmail.com

Organization name

ACADEMIA SINICA

Department

INSTITUTE OF PLANT AND MICROBIAL BIOLOGY