Schematic view of the alternative splicing library construction with amplification of RNA. I. Oligo dT containing T7 RNA Polymerase recognition site was used for first strand cDNA synthesis with Superscript II that adds cytosine residues after reaching the 5`end of mRNAs. II. This c-rich region serves as anchor for TS-oligo alignment, allowing further polymerization to the end of the oligo. III. Second strand cDNA synthesis using TS-oligo. IV. Amplification of mRNA using T7 RNA Polymerase. V. First strand cDNA synthesis using TS-oligo. VI. Second strand cDNA synthesis using oligodT. VII. Denaturation and renaturation resulting in the formation of heteroduplexes molecules by common exons complementarity. VIII. Single-stranded molecules degraded by Exonuclease (dotted line). IX. DpnII digestion resulting in small cohesive fragments. X. 25mer biotinilated random oligos coupled to streptavidin magnetic beads anneal to single-strand loops. XI. Coupling of specific adaptors to the cohesive ends of the captured heteroduplexes. XII. PCR amplification of fragments using adaptors specific oligos (double line).
Ferreira et al. BMC Genomics 2010 11(Suppl 5):S4 doi:10.1186/1471-2164-11-S5-S4