| Construction Protocol | The full-length cDNA library was constructed essentially as reported previously (Seki et al. (1998) Plant J. 15:707-720; Seki et al. (2002) Science 296:141-145). The cDNAs derived from male strobili of C. japonica are inserted into a modified pBluescript vector. Plasmid DNA was digested by PI-Sce I whose site is placed near the 3'-end of a cDNA insert and then sense strand RNA was synthesized by RNA polymerase. First strand cDNA was synthesized using an oligo dT primer with resriction site of Gsu I. RNA of double stranded DNA:RNA hybrids was degraded by alkaline treatment. Second strand was synthesized using a primer annealed to a contiguous region of the 5'-end of a cDNA. Purified double strand DNA was digested by Gsu I to remove a 3' poly(A) tail. This DNA fragment was used for sequencing. The subsequent procedure was performed using GS FLX Titanium Rapid Library Preparation Kit and Rapid Library MID Adaptors Kit. |
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