How do I prepare an RNA sequencing library?

The first step in preparing an RNA sequencing library is the removal of abundant transcripts. Most RNA molecules present in a cell are rRNA, and are typically not of interest. Thus, they should be removed prior to making a library from the RNA of interest. Globin RNA is typically removed as well from blood samples. Fragmentation can be used to do this, and fragments of a proper size for sequencing are produced by fragmentation of RNA prior to reverse transcription and cDNA synthesis. Another method to remove rRNA is through reverse transcription and second-strand cDNA synthesis. cDNA is produced from the RNA template by reverse transcriptase. The first strand is then produced into a double strand using DNA polymerase. After the removal of rRNA, end repair of the ds cDNA library is followed by ligation to adaptors. These adapters possess the full complement of sequencing primer hybridization sites. Thus, additional PCR steps are not required and the process is fully automatable. The library is then ready for sequencing and amplification.