GSM1621381: SS; Brassica juncea; RNA-Seq
Total RNA was extracted from the frozen tissue using TRIzol reagent and purified using oligo dT beads (TruSeq RNA Sample Preparation Kit, Illumina). The purified mRNA was fragmented at elevated temperature (90°C) in the presence of divalent cations and reverse transcribed with Superscript II Reverse Transcriptase (Invitrogen Life Technologies) by priming with random hexamers. Second strand cDNA was synthesized in the presence of DNA polymerase I and RNaseH. The cDNA was cleaned using Agencourt Ampure XP SPRI beads (Beckman Coulter). Illumina adapters were ligated to the cDNA molecules after end repair and addition of an ‘A’ base followed by SPRI clean-up. The resultant cDNA library was amplified using PCR for enrichment of adapter ligated fragments, quantified using a Nanodrop spectrophotometer (Thermo Scientific) and validated for quality with a Bioanalyzer (Agilent Technologies). It was then sequenced using the Illumina’s Hi-Seq 2000 platform. RNA libraries were prepared for sequencing using standard Illumina protocols
GEO Accession
GSM1621381