title: Illumina Genome Analyzer II paired end sequencing; RNA-seq of a P. infestans resistance segregating population with parents Desiree (cv) and breeding line SW93-1015
design: RNA-seq of a P. infestans resistance segregating population with parents Desiree (cv) and breeding line SW93-1015
library_construction_protocol: Potato plants were grown under controlled conditions with 16 h/8 h day and night regime with a fluorescent lamp light intensity of 200 μmol m-2 s-1. The temperature was set to 20°C and the relative humidity was 65%. Pots were circulated every week to avoid positional effects. All sampling was done before flowering and only fully expanded leaves from the middle part of the plants were used.  RNA was isolated by RNeasy Plant Mini Kit (Qiagen) Beads with oligo(dT) were used to isolate poly(A) mRNA. Fragmentation buffer was added for interrupting mRNA to short fragments. Taking these short fragments as templates, Random hexamerprimer were used to synthesize the first-strand cDNA. The second-strand cDNA was synthesized using buffer, dNTPs, RNase H and DNA polymerase I, respectively. Short fragments were purified with QiaQuick PCR extraction kit and resolved with EB buffer for end reparation and adding poly(A). After that, the short fragments were connected with sequencing adaptors. And, for amplification with PCR, we selected suitable fragments, as templates, with respect to the result of agarose gel electrophoresis.
attributes.tag: Experimental Factor: individual
attributes.value: L13