RNA extraction and library preparation for transcriptome analysis

Total RNA of roots with and without atrazine were extracted using the TRIzol Reagent (Invitrogen) and treated with DNase I according to manufacturer’s instructions. RNA quality was examined using 2% agarose gel and the concentration was determined using a Nanodrap spectrophotometer (NanoDrop, USA). Then, preparation of cDNA libraries for Illumina HiSeq 2500 sequencing was done using the Truseq^TM RNA sample prep kit, following the manufacturer’s instructions. The isolation of mRNA, fragment interruption, cDNA synthesis, addition of adapters, PCR amplification and RNASeq were performed by staff at Shanghai Majorbio Bio-Pharm Technology (Shanghai, China). Poly-A mRNA was isolated using Magnetic Oligo (dT) Beads, and then broken into small pieces using divalent cations under an elevated temperature. Then the double-stranded cDNA was synthesized using the SuperScript Double-Stranded cDNA Synthesis kit (Invitrogen, Camarillo, CA) with random hexamer primers (Illumina). The synthesized cDNA was subjected to end-repair and phosphorylation using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK. These repaired cDNA fragments were 3′ adenylated using Klenow Exo- (3′ to 5′ exo minus, Illumina). Illumina Paired-end adapters were ligated to the ends of these 3′-adenylated cDNA fragments. Fragments (200 bp ± 25 bp) were then separated by agarose gel electrophoresis and selected for PCR amplification as sequencing templates. Finally, the mRNA-seq library was constructed for sequencing on the Illumina HiSeqTM 2500 sequencing platform. cDNA library construction process was shown in Fig. 9.