GSM1421962: CT Endosperm 2 [RNA-seq]; Brassica rapa; RNA-Seq
All tissue samples harvested from control and treated plants were subjected to total RNA isolation using the Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Two independent RNA samples per every tissue were prepared. Total RNA was further purified and concentrated using the RNA Clean-Up and Concentration Kit (Norgen Biotek Corp., Ontario, Canada). The quality and concentration of every sample were quantified using the NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific Inc.) and the absorption ratios at 260/280 and 260/230 nm wavelengths. Agarose gel electrophoresis was performed to verify RNA sample integrity. The mRNA libraries were prepared from 16 samples of total RNA in 2 biological replicates according to the TruSeq RNA sample Prep v2 LS protocol (Illumina, San Diego, CA, U.S.A.). Briefly, mRNA was purified from the total RNA samples using poly-T oligo-attached magnetic beads followed by mRNA fragmentation, first- and second-strand cDNA synthesis. Later, the overhangs resulting from the fragmentation of double-stranded (ds) cDNA were repaired to form blunt ends. A single ‘A’ nucleotide was added to the 3΄ ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction. Multiple indexing adapters were ligated to the ends of ds cDNA to prepare them for hybridization onto a flow cell followed by a PCR amplification step. The libraries were quantified using the qPCR technique and analyzed on a Bioanalyzer 2100 (Agilent Technologies) using a DNA specific chip. Subsequently, the libraries were normalized and pooled together followed by flow-cell cluster generation using a cBot fully automated clonal cluster generation system for Illumina sequencing. Single-end multiplexed sequencing was done using the Illumina GAIIx platform with the total of 100 cycles.