Illumina HiSeq 2000 sequencing; RNA-seq of barley lines overexpressing or silencing HvPap-1 gene.
RNA-seq of barley lines overexpressing or silencing HvPap-1 gene.
Barley plants of Hordeum vulgare cv. Golden Promise were used. Grains were germinated in soil and grown at 22ºC under a 16 h light/8 h darkness photoperiod for 7 days in Sanyo MLR-350-H chambers. Barley transgenic lines overexpressing or silencing the HvPap-1 gene (OE Pap1 and KD Pap1, respectively) were generated using Agrobacterium-mediated gene transfer on immature embryos and homozygous material obtained by double haploid technology. Transgenic barley plants ubiquitously over-expressing the HvPap-1 gene were generated using p6U and p6d35s binary vectors. Silencing of the HvPap-1 gene was performed using the artificial microRNA (amiRNA) technology. Total RNA was extracted from frozen barley leaves by the phenol/chloroform method, followed by precipitation with 8 M LiCl  and digested with DNase (Promega).  Using poly-T oligo-attached magnetic beads, mRNAs were purified from the total RNA. Then, the mRNAs were fragmented and cDNA was synthesized using random hexamer-primers, DNA polymerase I and RNase H. The double-stranded cDNAs were purified with magnetic beads and ligated to adaptors for Illumina sequencing. The quality and quantity of the library was verified using an Agilent 2100 Bioanalyzer and an ABI StepOnePlus Real-Time PCR system, respectively.