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).