GSM1566892: B. juncea drought stressed (BDS) (UNPAIRED); Brassica juncea; RNA-Seq
Total RNA was isolated using GITC-based method from abiotic stress treated and untreated whole seedlings, independently for each time point. Extracted RNA was quantified using spectrophotometer and an aliquot of heat denatured RNA was electrophoresed on denaturing agarose gel to check its integrity. RNA extracted from two different time points were pooled in equimolar amounts and three RNA-Seq libraries- BC (control seedlings), BDS (drought stressed seedlings) and BHS (high temperature stressed) were prepared utilizing NEBNext RNA-Seq library preparation Master Mix Set for Illumina procured from NEB, USA. Poly A+ RNA was isolated from 10 µg of total RNA using Sera-Mag beads (GE Healthcare, UK) and fragmented chemically at high temperature. Fragmented RNA was qualitatively and quantitatively checked on Bioanalyzer (Agilent, USA). 250 ng of fragmented RNA was used for first strand reverse transcription using random primers followed by second strand synthesis. The ends of double stranded cDNA were repaired and mono-adenylated. Paired end adapters were ligated using Rapid T4 DNA ligase and then size fractionated. Approximately, 350 bp size region was eluted and PCR amplified for 12 cycles. The quality and quantity of prepared libraries was evaluated utilizing Bioanalyzer (Agilent, USA) Ultra-deep parallel sequencing was performed using Illumina Genome Analyzer IIx at University of Delhi South Campus, Delhi, India according to manufacturer’s instructions.