Physcomitrella patens Gransden gametophores 5UTR RNAseq
For cDNA synthesis a CAP targeting strategy was used. For this purpose, the juvenile gametophores and the protonema poly(A)+ RNAs were first incubated with CIP (calf intestine phosphatase). After the removal of CIP, the dephosphorylated RNAs were then treated with TAP (tobacco acid pyrophosphatase). To the 5´-phosphate of the decapped mRNAs a short RNA oligonucleotide was ligated. First-strand cDNA synthesis was then performed using a N6 randomized adapter primer and M-MLV-RNase H- reverse transcriptase. The resulting cDNAs were finally amplified with 16 cycles of LA-PCR. For sequencing with the Solexa platform the cDNAs in the size range of 250   450 bp were eluted from preparative agarose gels. Aliquots of the size fractionated cDNAs were analyzed on a 1,5% agarose gel.