Small RNA sequencing library preparation using NEBNext® begins with either total RNA or purified small RNA. This high-yield method is suitable for methylated small RNAs (e.g. piRNAs) as well as unmethylated small RNAs.
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′ adaptors that are ligated to small RNA molecules, the RT primer also hybridizes to any excess 3′ adaptors 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 dNTPs. Reverse transcribed molecules that contain both adaptor sequences are then substrates for amplification by PCR. Libraries are characterized using gel electrophoresis with a Bioanalyzer® or similar method, and the amplified library is isolated for use in Illumina® sequencing.
NEBNext Small RNA kits include 12 or 48 index primers for multiplexing:
NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (Set 1)
NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (Set 2)
NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (Index Primers 1-48)