Overview of RNA-Seq. First, RNA is extracted from the biological material of choice (e.g., cells, tissues). Second, subsets of RNA molecules are isolated using a specific protocol, such as the poly-A selection protocol to enrich for polyadenylated transcripts or a ribo-depletion protocol to remove ribosomal RNAs. Next, the RNA is converted to complementary DNA (cDNA) by reverse transcription and sequencing adaptors are ligated to the ends of the cDNA fragments. Following amplification by PCR, the RNA-Seq library is ready for sequencing.