Adenylation of 3’ adapter using T4 RNA ligase 1. (A) Effect of PEG8000 concentration on adenylation efficiency. A synthetic oligo BL1 mimicking the first small RNA cloning linker reported by Lau et al. (2001) was adenylated overnight with 1 U/μL T4 RNA ligase at various PEG concentration. Non-adenylated oligo as the negative control (NC) is loaded on the left lane. (B) Effect of temperature and 5’ nucleotide composition on adenylation efficiency. Oligos were adenylated overnight in the presence of 20% PEG8000 at various temperatures. (C) Impact of oligo concentration on adenylation efficiency. Substrates with different concentrations were adenylated overnight with 20% PEG8000 at room temperature. All adenylation products were analyzed on the 20% denatured PAGE, stained with SYBR-Gold and photograph under UV.