RNA-seq analysis to investigate the downstream genes of MeCBF4 in Arabidopsis
Expression profile of wild-type and transgenic A. thaliana was carried out by RNA-seq. Three biological replicates were used for each genotype under normal growth conditions. Total RNA was extracted from samples with CTAB solution. RNA samples are first treated with DNase I to degrade any possible DNA contamination. The mRNA is fragmented into short fragments (about 200 bp) and enriched by PCR amplification to construct cDNA library. The library products were sequenced via Illumina HiSeqTM 2000. Clean reads were obtained through filtering low quality reads and adaptor sequences in raw reads harvested from base calling and mapped to the A. thaliana reference genome database (http://www.arabidopsis.org/) through SOAPaligner/SOAP2 [36]. The gene expression level is calculated by using RPKM method (Reads Per kb per Million reads) . Corrected P-value of 0.005 and log2 (fold change) of 1 were set as the threshold for significantly differential expression. The function of genes was annotated by using BLASTx with an E-value threshold of 10-5to the NCBI nr, Swiss-Prot, KEGG, COG, and A. thaliana Genome databases.