Fig. 3.

Diagram of library preparation procedure. Membrane-isolated RNA is first polyadenylated (1). The RNA is then reverse transcribed, however, the enzyme frequently fails to read through the cross-link site, leading to termination followed by the addition of non-templated nucleotides bound by the smRNA oligonucleotide (2). Template switching and extension by reverse transcriptase completes the addition of an adaptor to the 3’ end of the first-strand cDNA (3). In the final step, PCR amplification is used to add full-length Illumina adaptors to the cDNA ends (4).