RNA sequencing of rice genotype Sahabhagidhan: after 24hrs of application of chilling stress
The rice genotype (Sahabhagidhan) was grown in pots under uniform growth conditions till 15days. The pots were exposed to cold stress condition using a Conviron PGV36 walk-in growth chamber. The light intensity was 800 µ moles s-1 m-2 at 60 cm above the floor while the relative humidity ranged from 75-85% with 12-h photoperiod. Leaf samples of the genotype were collected after 24h of exposure to chilling stress at 4 degree Celsius. Total RNA was extracted separately from approximately 100 mg leaf from each sample using XcelGen total RNA isolation kit (Xcelris Genomics, India) as per manufacturer’s instructions. The yield and purity of RNA were evaluated by recording absorbance at 260 and 280 nm on Nanodrop 8000 spectrophotometer (Thermoscientific). Further, the integrity of RNA was checked using RNA 6000 Nano LabChip using Agilent Bioanalyzer 2100 (Agilent Technologies, Germany). The pair end sequencing libraries for RNA-seq were prepared using illumina TruSeq® RNA Library Preparation Kit as per manufacturer's protocol (illumina®, San Diego, CA). Library quality control and quantification were performed on Caliper LabChip GX using HT DNA High Sensitivity Assay Kit. RNA-Seq was performed using illumina HiSeq2000 (illumina®, San Diego, CA) as per manufacturer's protocol to generate 30 million 2x100 bp reads for each 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).
Rice genotype Sahabhagidhan: after 24hrs of application of chilling stress