We developed PolyA-seq, a strand-specific and quantitative method for high-throughput sequencing of 39 ends of polyadenylated transcripts, and used it to globally map polyadenylation (polyA) sites in 24 matched tissues in human, rhesus, dog, mouse, and rat. We show that PolyA-seq is as accurate as existing RNA sequencing (RNA-seq) approaches for digital gene expression (DGE), enabling simultaneous mapping of polyA sites and quantitative measurement of their usage. In human, we confirmed 158,533 known sites and discovered 280,857 novel sites (FDR < 2.5%). On average 10% of novel human sites were also detected in matched tissues in other species. Most novel sites represent uncharacterized alternative polyA events and extensions of known transcripts in human and mouse, but primarily delineate novel transcripts in the other three species. A total of 69.1% of known human genes that we detected have multiple polyA sites in their 39UTRs, with 49.3% having three or more. We also detected polyadenylation of noncoding and antisense transcripts, including constitutive and tissue-specific primary microRNAs. The canonical polyA signal was strongly enriched and positionally conserved in all species. In general, usage of polyA sites is more similar within the same tissues across different species than within a species. These quantitative maps of polyA usage in evolutionarily and functionally related samples constitute a resource for understanding the regulatory mechanisms underlying alternative polyadenylation.[Supplemental material is available for this article.]Sequencing of mRNA and noncoding RNA has made important contributions to our understanding of biology and disease, with numerous implications for diagnostics and therapeutics. As an outcome of rapidly expanding sequencing capabilities, recently described methods produce comprehensive representations of the transcriptome (Mortazavi et al. 2008;Armour et al. 2009;Wang et al. 2009;Levin et al. 2010) and have been used to discover and monitor alternative splicing (Sultan et al. 2008;Wang et al. 2008;Wilhelm et al. 2008), as well as gene expression (Marioni et al. 2008) and its underlying regulatory genetic variation (Montgomery et al. 2010;Pickrell et al. 2010). While transcriptome sequencing studies continue to focus on gene expression and RNA processing, mapping of polyA sites has received considerably less attention, despite evidence suggesting that alternative polyadenylation is common in metazoans (Lee et al. 2007;Ozsolak et al. 2010) and contributes to phenotypic variation and disease. Avoidance of microRNA regulation via alternative polyA sites, for example, plays a role in development (Mangone et al. 2010;Thomsen et al. 2010;Jan et al. 2011) and cancer Mayr and Bartel 2009). Furthermore, extensive usage of tissue-specific sites, some of which are associated with cis-regulatory elements, suggests that alternative polyadenylation is tightly regulated (Proudfoot et al. 2002) and has important physiological implications. Lastly, the 39UTRs of some genes are expressed ...

A technique is described for specific, sensitive, quantitative, and rapid detection of biological targets by using superparamagnetic nanoparticles and a ''microscope'' based on a high-transition temperature dc superconducting quantum interference device (SQUID). In this technique, a mylar film to which the targets have been bound is placed on the microscope. The film, at room temperature and atmospheric pressure, is typically 40 m from the SQUID, which is at 77 K in a vacuum. A suspension of magnetic nanoparticles carrying antibodies directed against the target is added to the mixture in the well, and 1-s pulses of magnetic field are applied parallel to the SQUID. In the presence of this aligning field the nanoparticles develop a net magnetization, which relaxes when the field is turned off. Unbound nanoparticles relax rapidly by Brownian rotation and contribute no measurable signal. Nanoparticles that are bound to the target on the film are immobilized and undergo Né el relaxation, producing a slowly decaying magnetic flux, which is detected by the SQUID. The ability to distinguish between bound and unbound labels allows one to run homogeneous assays, which do not require separation and removal of unbound magnetic particles. The technique has been demonstrated with a model system of liposomes carrying the FLAG epitope. The SQUID microscope requires no more than (5 ؎ 2) ؋ 10 4 magnetic nanoparticles to register a reproducible signal.