Deep sequencing of short capped RNAs reveals novel families of noncoding RNAs.

In eukaryotes, capped RNAs include long transcripts such as messenger RNAs and long noncoding RNAs, as well as shorter transcripts such as spliceosomal RNAs, small nucleolar RNAs, and enhancer RNAs. Long capped transcripts can be profiled using cap analysis gene expression (CAGE) sequencing and other methods. Here, we describe a sequencing library preparation protocol for short capped RNAs, apply it to a differentiation time course of the human cell line THP-1, and systematically compare the landscape of short capped RNAs to that of long capped RNAs.

Analysis of RNA decay factor mediated RNA stability contributions on RNA abundance.

Background: Histone epigenome data determined by chromatin immunoprecipitation sequencing (ChIP-seq) is used in identifying transcript regions and estimating expression levels. However, this estimation does not always correlate with eventual RNA expression levels measured by RNA sequencing (RNA-seq). Part of the inconsistency may arise from the variance in RNA stability, where the transcripts that are more or less abundant than predicted RNA expression from histone epigenome data are inferred to be more or less stable.

Genome-wide analysis of long noncoding RNA turnover.

Genome-wide analysis for determining RNA turnover is an advanced method in RNA biology that examines the specific half-life of nuclear noncoding RNA (ncRNA). In particular, a pulse-labeling method using uridine analogs enables the determination of RNA stability under physiologically undisturbed conditions. The technique involves pulse labeling of endogenous RNAs in mammalian cells with 5'-bromo-uridine (BrU), followed by measuring the chronological decrease of BrU-labeled RNAs using deep sequencing.

RNA sequencing: from sample preparation to analysis.

The introduction of RNA sequencing (RNA-seq), as a direct result of rapid progression of next-generation sequencing technologies, has revolutionized the world of transcriptomics. It enables quantification of the complete set of RNA with all its isoforms in a given cell, in far greater accuracy than before. Here, we provide a step-by-step guide to the sample preparation in RNA-seq and an overview of the bioinformatic analysis that is required following the cDNA sequencing.