Monitoring comparative transcriptional changes in a susceptible and tolerant landrace of cassava infected with South African cassava mosaic virus using next- generation sequencing
cDNA library preparation and sequencing cDNA libraries were generated at the Functional Genomics Center UNI ETH Zurich, Switzerland. Briefly, 12ug of total RNA for each sample was used to generate cDNA libraries. RNA was fragmented and subjected to hybridization and ligation using the SOLiD Total RNA-Seq Kit (Applied Biosystems) according to the manufacturer's instructions. cDNAs were selected by size on a polyacrylamide gel before and after the library amplification. A total of 12 libraries were multiplexed using the SOLiD RNA Barcoding Kit (Applied Biosystems) and pooled in an equimolar ratio. The samples were then diluted and used for emulsion PCR. Beads containing a multiplex of 12 samples were deposited onto a single flow cell. Libraries were sequenced operating on 50bp forward and 35 bp reverse paired-end sequencing chemistry on the ABI SOLiD V4 system.