Transcriptome Analysis of Rice Seedlings under Phosphate Deficiency

RNA was extracted from 0.1 g of frozen aerial parts of rice seedlings grown under 24-h Pi deficiency or control conditions using a RNeasy Plant Mini kit (Qiagen). The RNA-Seq libraries were prepared with a TruSeq Stranded mRNAseq Sample Prep kit (Illumina). The libraries were quantitated by qPCR and sequenced for 101 cycles from one end of the fragments on a HiSeq2500 using a HiSeq SBS sequencing kit version 4. Fastq files were generated and demultiplexed with the bcl2fastq v2.17.1.14 Conversion Software (Illumina). Illumina reads of all samples have been submitted to the Sequence Read Archive at the NCBI under accession number SRP102661. RNA-Seq reads for each sample were mapped to the reference genome (MSU Rice Genome Annotation Release 7.1) using the Bowtie2 tool (Langmead and Salzberg, 2012 (link)). To quantify transcript abundance, the Cuffdiff tool was applied to obtain fragments per kilobase of transcript per million mapped reads (FPKM) (Trapnell et al., 2012 (link)). Differentially expressed genes were identified using the DESeq2 package (FDR<0.001) (Love et al., 2014 (link)) and the normalized rLog (regularized Log-transformation) values of selected genes (n=2002) with FDR<0.001 obtained from DESeq2 were used for clustering. The fuzzy k-means clustering was done with the Aerie tool (Gasch and Eisen, 2002 (link)).