Mouse wild type spleen EDGE BiologicalReplicate_3
Double stranded cDNA synthesis is performed using paramagnetic oligo-dT beads to capture polyadenylated RNA. Next, each cDNA molecule is anchored by NlaIII restriction cleavage that exposes the 3prime-most CATG site within the transcript. Following this, the EDGE_Rev adaptor carrying an EcoP15I recognition site is ligated, and the resulting molecule is tagged by EcoP15I restriction digest, generating a 27 bp sequence tag. The sticky end is ligated to the EDGE_For adaptor. Finally, a 15-cycle PCR amplification using adaptor-specific primers is performed to add on the additional sequence required to complete the EDGE_For adaptor and to enrich for the desired final product.