title: Sequencing of Caragana Intermedia's transcriptome
design: Solexa cDNA library construction and sequencing  PolyA-containing mRNAs were purified using Sera-mag Magnetic oligo-dT-attached beads (Illumina) from 10 ug of total RNAs after RNA extraction. To avoid priming bias when synthesizing cDNA, the purified mRNA was first fragmented into small pieces (100-400bp) using divalent cations at 94°C for exactly 5 minutes. The cleaved short mRNA fragments were used as templates to synthesize the first-strand cDNA using DNA reverse transcriptase and random hexamers (N6) primers (Invitrogen Inc). This was followed by second-strand cDNA synthesis using buffer, dNTPs, RNase H and DNA polymerase I using the SuperScript Double-Stranded cDNA Synthesis kit (Invitrogen, Camarillo, CA).    The synthesized cDNA was subjected to end-repair and phosphorylation using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK. These repaired cDNA fragments were 3' adenylated using Klenow Exo- (3' to 5' exo minus, Illumina). Illumina Paired-end adapters were ligated to the ends of these 3'-adenylated cDNA fragments.  To select a size range of templates for downstream enrichment, the products of ligation reaction were subsequently purified on a 2% TAE-agarose gel (Certified Low-Range Ultra Agarose, Biorad) and amplified through PCR to create the final cDNA libraries. Fifteen rounds of PCR amplification were performed to enrich the purified cDNA template using PCR Primer PE 1.0 and PCR Primer PE 2.0 (Illumina)] with Phusion DNA Polymerase. The amplification conditions were as following: 94? 30s, 60? 30s, 72? 3 min. The cDNA library was constructed with a fragment length range of 200 bp (±25 bp). The finally collected double-strain cDNA (10ug, 1ug/ul concentration) was send to Beijing Genomics of Institute (BIG). Illumina sequencing using the GAII platform was performed at Beijing Genomics Institute (BGI)-Shenzhen, Shenzhen, China  http://www.genomics.cn/index.php webcite according to the manufacturer's instructions (Illumina, San Diego, CA). Finally, after validating on an Agilent Technologies 2100 Bioanalyzer using the Agilent DNA 1000 chip kit, the cDNA library was sequenced on a PE flow cell using Illumina Genome Analyzer IIx, and the workflow was as follows: template hybridization, isothermal amplification, linearization, blocking, sequencing primer hybridization, and sequencing on the sequencer for Read 1. After completion of the first read, the templates can be regenerated in situ to enable a second 75 bp read from the opposite end of the fragments, i.e., the newly sequenced strands are stripped off and the complementary strands are bridge amplified to form clusters. Once the original templates are cleaved and removed, the reverse strands undergo sequencing-by-synthesis, generating 36,991,854 sequencing reads with 75-mer length.