Enables preparation of a variety of RNA species, using total RNA or purified small RNA as input.
Takes advantage of the natural structure common to most known microRNA molecules.
Protocol includes adapter ligation, reverse transcription, PCR amplification, and pooled gel purification to generate a library product.
RNA 3' adapter is specifically modified to target microRNAs and other small RNAs that have a 3' hydroxyl group resulting from enzymatic cleavage by Dicer or other RNA processing enzymes.
Adapters are ligated to each end of the RNA molecule and an RT reaction is used to create single-stranded cDNA.
cDNA is PCR amplified using a common primer and a primer containing one of 48 index sequences.
Introduction of the index sequence at the PCR step separates the indexes from the RNA ligation reaction, allowing for the indexes to be read using a second read and significantly reducing bias compared to designs which include the index within the first read.
Allows the use of 48 different index tags to make use of the Illumina multiplexing capability for analyzing directional and small RNA samples.
Employs six-base indices to distinguish different samples from one another in a single lane of a flow cell.