Single Cell Micro RNA Sequencing Library Preparation.

Micro RNAs represent important post-transcriptional regulators in health and are involved in the onset of many diseases. Therefore, the further characterization of physiological miRNA functions is an important basic research question, and miRNAs even have high potential as biomarkers both for prognosis and diagnosis. In order to exploit this potential, it is mandatory to accurately quantify the miRNA expression not only in bulk but also on the single-cell level. Here, we describe a protocol, which facilitates miRNA sequencing library preparation of very low input samples, single cells, and even clinical samples such as circulating tumor cells. The protocol can be combined with different single-cell isolation methods (e.g., micromanipulation and FACS sorting). After cell lysis, sequencing adapters are ligated to the miRNAs, other ncRNA species, and adapter dimers are reduced by exonuclease digest, the miRNA library is reverse transcribed, amplified, and purified. Furthermore, quality controls are described to select only high-quality samples for sequencing.