Fig. 2.

Optimization of experimental procedures. A The effect of reaction time on the normal and 5′ dU adapter cleavage. The figure shows the cleavage of both normal (lane 1–2) and dU adapter (lane 3–7) with USER II at different reaction time. B The effect of column purification on the USER II cleavage after the ligation step. The 106-nt ligated product bands without USER II cleavage before and after column purification (lanes 1 and 3) are defined as a reference for calculation. Equation 3 is used for calculating the reduction rate of ligated product (see the “Methods” section). C The effect of column purification on cleaved adapters removal after USER II treatment. The cleaved adapters generated from the USER II treatment (lane 3) are used as the reference to calculate the reduction rate of the cleaved adapters after column purification followed by the USER II step (lane 4). The N40+22 DNA only (62 nt in lane 1) and dU adapter only (44 nt in lane 2) are used as size references. Equation 5 is used for calculating the reduction rate of cleaved adapters (see the “Methods” section). D, E The effect of normal adapter and dU adapter on the PCR amplification. Agarose gel image of the PCR result is shown in panel E. PCR products of the library generated with 8, 11, 13 and 17 PCR cycles using a normal adapter (Norm) and 5′ dU adapter. Comparison of the relative quantity of PCR product is shown in panel F. 8 cycles of PCR product with normal adapter is defined as 1. The DNA size marker indicates the fragment size. The errors showed are standard deviation. nt=nucleotide. Three biological replicates are performed in A–C and two biological replicates in D–E. Raw data values are provided in the Additional file 8