Solexa sequencing of AS library
In order to obtain cDNA fragments which cover the complete transcripts, prior to cDNA synthesis the RNAs were fragmented by incubating at 95 degree Celsius for 5 minutes. The RNA fragments were then poly(A)- tailed using poly(A) polymerase followed by ligation of a RNA adapter to the 5'-phosphate of the RNA fragments. First-strand cDNA synthesis was then performed using an oligo(dT)-adapter primer and MMLV reverse transcriptase. The resulting cDNAs were PCR-amplified to about 20 ng/*l using a high fidelity DNA polymerase. The primers used for PCR amplification were designed for amplicon sequencing according to the instructions of Illumina/Solexa. The following adapter sequences flank the cDNA inserts: 5'- end (53 bases) with Tag sequence 5'- AATGATACGGCGACCACCGACAGGTT-CAGAGTTCTACAGTCCGACGATCNNNN-3' 3'-end (45 bases): 5'- CAAGCAGAAGACGGCATACGATTTTTTTTTTTTTTTTTTTTTTTT-3'