Expression analysis by RNA sequencing and qRT-PCR
RNA isolation and sequencing
Total RNA was isolated from leaf sample from five-week oldTaHSFA6b overexpressing transgenic plants and wild type plants using the RNEasy plant RNA isolation kit with on column DNase digestion, as per manufacture’s instruction (Qiagen, Germany). For this two overexpressing lines were used along with 2 replicates of wild type plants. High quality RNA samples were sent to Bionivid Technology Private Limited (Bengaluru, India) for RNA-sequencing by Illumina HiSeq platform with a read length of 150bp.
Quality control of the all the reads was done using NGSQC Tool kit (Patel et al. 2012) and reads having a Phred score >Q30 were selected for further analysis. For alignment of reads and transcript identification, the reference genome of barley was downloaded from ENSEMBLE (plants/release40/gff3/Hordeum-vulgare). For alignment of reads on the reference genome of barley TopHat pipeline was used (Trapnell et al. 2009). Cuffdiff and Cufflink pipelines were used for identification of differentially expressed transcripts in transgenic lines and wild type by using default parameters (Trapnell et al. 2009). Transcripts with fold change log2 ratio≥2 were considered as differentially expressed transcripts. Unsubstantiated hierarchical clustering of differentially expressed genes was performed by using Cluster 3.0 and visualized by using Java Tree View. Gene ontology and KEGG pathways that contained expressed transcripts were identified by using DAVID Functional Annotation Tool (DAVID Bioinformatics Resources 6.8, NIAID/NIH) (Hua ng et al. 2009).