RNA-seq of Schizosaccharomyces pombe : antisense transcription
10_g total RNA was used as starting material for strand-specific RNA-seq library preparation. Poly(A)+ RNA was enriched by two rounds of poly(dT) Sera-Mag magnetic beads purification. Enriched poly(A)+ RNA was then fragmented to an average size of ~200nt. Fragmented RNA was 3Ő de-phosphorylated with Antartic phosphatase and 5Ő phosphorylated with polynucleotide kinase this treatment prepares RNA fragments for subsequent ligation of Illumina RNA adaptors to their 5Ő and 3Ő ends using a 3Ő RNA ligase and a T4 RNA ligase, respectively. First strand cDNA was produced using a primer specific for the Illumina 3Ő adaptor. The library was finally amplified with 15 cycles of PCR using primers specific for the Illumina adaptors and purified using SPRI-beads (Agencourt, Beckman Coulter).