Library preparation of small RNA begins with either total RNA or purified small RNA.
The first step is to ligate an adaptor to 3′ end of the single-stranded RNA. The next step is the introduction of the RT primer. In addition to hybridizing to the 3′ adaptor that are ligated to small RNA molecules, the RT primer also hybridizes to any excess 3′ adaptor from the first step, thus forming a double-stranded molecule that is not accessible to ligation of the 5′ adaptor in the next step. This important step prevents the formation of contaminating adaptor dimers. After ligation of the 5’ adaptors, the small RNA becomes the template for the synthesis of one complementary strand of DNA, in the presence of a reverse transcriptase, primers and deoxynucleotide triphosphates (dNTPs) (A, T, G, C). Reverse transcribed molecules that contain both adaptor sequences are then substrates for amplification by the polymerase chain reaction (PCR) amplification. Libraries are characterized using gel electrophoresis, and the amplified cDNA construct of interest is isolated for use in Illumina® and SOLiD™ platforms.