Illumina HiSeq 2500 sequencing; sRNA sequencing of TRV-infected Nicotiana benthamiana 16c at 29oC
sRNA sequencing of TRV-infected Nicotiana benthamiana 16c at 29oC
Samples were collected from upper systemic leaves of TRVs infected plants at one week post infection and three weeks post infection. The collected samples were flash frozen in liquid nitrogen. Plants were grown in Levington F2+S professional growth compost in a controlled growth chamber (SANYO/Panasonic) at 22°C with 16-h light and 8-h dark periods for 4 weeks. Plants were infected at 4 weeks of age. After viral inoculations, the infected plants were maintained at 22°C for 2 days and then shifted to 29°C for long term. Total nucleic acid (TNA) extracts were prepared from leaf tissue by phenol-chloroform extraction as described in White and Kaper, 1989. Small RNA libraries were prepared from 1 μg of TNA using the Illumina TruSeq Small RNA Library Prep Kit and sequenced on the Illumina HiSeq 2500 platform with 50-base single end reads at Edinburgh Genomics (Edinburgh, UK).