Single-Cell RNAseq Analysis of lncRNAs.

Mammalian genomes are pervasively transcribed and a small fraction of RNAs produced codify for proteins. The importance of noncoding RNAs for the maintenance of cell functions is well known (e.g., rRNAs, tRNAs), but only recently it was first demonstrated the involvement of microRNAs (miRNAs) in posttranscriptional regulation and then the activity of long noncoding RNAs (lncRNAs) in the regulation of miRNAs, DNA structure and protein function. LncRNAs have an expression more cell specific than other RNAs and basing on their subcellular localization exert different functions. In this book chapter we consider different protocols to evaluate the expression of lncRNAs at the single cell level using genome-wide approaches. We considered the skeletal muscle as example because the most abundant tissue in mammals involved in the regulation of metabolism and body movement. We firstly described how to isolate the smallest complete contractile system responsible for muscle metabolic and contractile traits (myofibers). We considered how to separate long and short RNAs to allow the sequencing of the full-length transcript using the SMART technique for the retrotranscription. Because of myofibers are multinucleated cells and because of it is better to perform single cell sequencing on fresh tissues we described the single-nucleus sequencing that can be applied to frozen tissues. The chapter concludes with a description of bioinformatics approaches to evaluate differential expression from single-cell or single-nucleus RNA sequencing.