Figure 1. Schematic representation of sequencing library preparation using heptamer primers based amplification, DP-seq.

(a) Step 1: Primer selection was based on identifying potential primer-binding sites that were less likely to form secondary structures and resided upstream to the unique regions on the mouse transcriptome. Step 2: targeted cDNA amplification. A Standard cDNA library was prepared and the primers selected from Step 1 were annealed to the single stranded cDNA library and were extended and amplified as indicated. Step 3: Library preparation. Illumina paired end adaptors were ligated to the ends of the amplicon library and the correct orientation of adaptors were selected. The library was further amplified using Illumina's paired end adaptor primers and were size selected for synthesis-based sequencing. (b) Expression profiles of genes responding to graded activation of the Activin A/TGFβ signaling pathway in mouse embryoid bodies at day 4. Quantitative RT-PCR data was normalized with respect to untreated serum-free media controls. (c) The fidelity of amplification of the cDNA library using heptamer primers. Fold changes observed in 11 genes (from part (b), Afp and Cer1) across different dosages of Activin A showed perfect agreement with quantitative RT-PCR performed on cDNA (R² = 0.94; n = 45). (d) Distribution of reads on the mouse genome.