The ability to profile mRNAs in single cells has transformed the molecular identification and the characterization of cell types, states and rare cellular phenotypes. However, current single-cell methods are restricted to long RNAs, primarily mRNAs. Several classes of small RNAs exist in mammalian cells, including microRNAs (miRNAs), and RNA fragments derived from tRNAs (tsRNAs, tRNA-derived small RNAs) and snoRNAs (sdRNAs, sno-derived RNAs), some of which have been shown to have tissue, cancer-type or cell-state-specific expression. Profiling of small RNAs has required large amounts of sample, but a more comprehensive understanding of their roles necessitates analysis of their expression patterns in single cells.
Here researchers from the Ludwig Institute for Cancer Research describe a method for sequencing the small-RNA transcriptome in single cells. They constructed libraries by ligating adapters to all RNA species harboring 5′ phosphate and 3′ hydroxyl groups regardless of their size. Masking oligonucleotides were designed to evade highly abundant 5.8S rRNA. Also, an enzymatic digestion step was applied to reduce the formation of adaptor dimers. Unique molecular identifiers (UMIs) were added to the 5′ adapters to counteract PCR stochasticity and to enable RNA molecule counting. No experimental size-selection step was used (simplifying automation) and small RNAs were identified computationally.
Schematic outline of the single-cell small-RNA library preparation
Single cells were lysed directly in the preparation tube and 5.8S rRNA was masked for adapter ligation immediately by a complementary oligo. Then, the 3′ adapter was ligated to the small RNAs and the free adapter was removed enzymatically prior to the 5′ adapter ligation. The 5′ adapter contains a unique molecular identiﬁer (UMI) to remove PCR replicates. Sequences required for Illumina cluster generation and sample indexing were added through two rounds of PCR ampliﬁcations.
“Our knowledge of the function of short RNA molecules is quite general. We have a picture of the general mechanisms, but it is less clear what specific role these molecules play in different types of cells or diseases,” explained senior study investigator Rickard Sandberg, Ph.D., associate professor in the department of cell and molecular biology at the Karolinska Institute and an affiliated member of the Ludwig Cancer Research in Stockholm. – Genetic Engineering News