Compositions and Methods for Constructing cDNA Libraries that Allow for Mapping the 5' and 3' Ends of RNAs

摘要

This disclosure provides methods and compositions for preparing and constructing cDNA libraries.

主权项

1. A method of optimizing the preparation of RNA molecules from a biological sample for sequencing, comprising of the steps of:
providing a biological sample that comprises RNA molecules; ligating a DNA adaptor to the 3′ end of the RNA molecules, wherein the ligating is performed under one or more conditions that optimize the ligation reaction; reverse transcribing the RNA molecules using a unique DNA primer under one or more conditions that optimize the reverse transcription reaction to produce single-stranded cDNA molecules; purifying the cDNA molecules under one or more conditions that limit loss of the single-stranded cDNA molecules; circularizing the purified cDNA molecules under one or more conditions that optimize the circularization reaction; amplifying the circularized cDNA molecules under one or more conditions that optimize the amplification reaction, thereby preparing RNA molecules from a biological sample for sequencing.