Massively parallel digital transcriptional profiling of single cells.

Characterizing the transcriptome of individual cells is fundamental to understanding complex biological systems. We describe a droplet-based system that enables 3' mRNA counting of tens of thousands of single cells per sample. Cell encapsulation, of up to 8 samples at a time, takes place in ∼6 min, with ∼50% cell capture efficiency.

Haplotyping germline and cancer genomes with high-throughput linked-read sequencing.

Haplotyping of human chromosomes is a prerequisite for cataloguing the full repertoire of genetic variation. We present a microfluidics-based, linked-read sequencing technology that can phase and haplotype germline and cancer genomes using nanograms of input DNA. This high-throughput platform prepares barcoded libraries for short-read sequencing and computationally reconstructs long-range haplotype and structural variant information.

Dicer-microRNA-Myc circuit promotes transcription of hundreds of long noncoding RNAs.

Long noncoding RNAs (lncRNAs) are important regulators of cell fate, yet little is known about mechanisms controlling lncRNA expression. Here we show that transcription is quantitatively different for lncRNAs and mRNAs--as revealed by deficiency of Dicer (Dcr), a key RNase that generates microRNAs (miRNAs). Dcr loss in mouse embryonic stem cells led unexpectedly to decreased levels of hundreds of lncRNAs.

Control of somatic tissue differentiation by the long non-coding RNA TINCR.

Several of the thousands of human long non-coding RNAs (lncRNAs) have been functionally characterized; however, potential roles for lncRNAs in somatic tissue differentiation remain poorly understood. Here we show that a 3.7-kilobase lncRNA, terminal differentiation-induced ncRNA (TINCR), controls human epidermal differentiation by a post-transcriptional mechanism.

Promoters recognized by forkhead proteins exist for individual 21U-RNAs.

C. elegans 21U-RNAs are equivalent to the piRNAs discovered in other metazoans and have important roles in gametogenesis and transposon control. The biogenesis and molecular function of 21U-RNAs and piRNAs are poorly understood.

Suppression of progenitor differentiation requires the long noncoding RNA ANCR.

Long noncoding RNAs (lncRNAs) regulate diverse processes, yet a potential role for lncRNAs in maintaining the undifferentiated state in somatic tissue progenitor cells remains uncharacterized. We used transcriptome sequencing and tiling arrays to compare lncRNA expression in epidermal progenitor populations versus differentiating cells. We identified ANCR (anti-differentiation ncRNA) as an 855-base-pair lncRNA down-regulated during differentiation.

Genome-wide impact of a recently expanded microRNA cluster in mouse.

Variations in microRNA (miRNA) gene and/or target repertoire are likely to be key drivers of phenotypic differences between species. To better understand these changes, we developed a computational method that identifies signatures of species-specific target site gain and loss associated with miRNA acquisition. Interestingly, several of the miRNAs implicated in mouse 3' UTR evolution derive from a single rapidly expanded rodent-specific miRNA cluster.

MicroRNAs can generate thresholds in target gene expression.

MicroRNAs (miRNAs) are short, highly conserved noncoding RNA molecules that repress gene expression in a sequence-dependent manner. We performed single-cell measurements using quantitative fluorescence microscopy and flow cytometry to monitor a target gene's protein expression in the presence and absence of regulation by miRNA. We find that although the average level of repression is modest, in agreement with previous population-based measurements, the repression among individual cells varies dramatically.

A latent pro-survival function for the mir-290-295 cluster in mouse embryonic stem cells.

MicroRNAs (miRNAs) post-transcriptionally regulate the expression of thousands of distinct mRNAs. While some regulatory interactions help to maintain basal cellular functions, others are likely relevant in more specific settings, such as response to stress. Here we describe such a role for the mir-290-295 cluster, the dominant miRNA cluster in mouse embryonic stem cells (mESCs).

Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without mature microRNAs.

MicroRNAs (miRNAs) are 19-22-nucleotide noncoding RNAs that post-transcriptionally regulate mRNA targets. We have identified endogenous miRNA binding sites in mouse embryonic stem cells (mESCs), by performing photo-cross-linking immunoprecipitation using antibodies to Argonaute (Ago2) followed by deep sequencing of RNAs (CLIP-seq). We also performed CLIP-seq in Dicer⁻/⁻ mESCs that lack mature miRNAs, allowing us to define whether the association of Ago2 with the identified sites was miRNA dependent.

Noncoding RNA landmarks of pluripotency and reprogramming.

Noncoding RNAs have emerged as important determinants of pluripotency and reprogramming. In this issue, Kosik and colleagues (Neveu et al., 2010) now provide a detailed map of microRNA expression patterns to infer the biological states of embryonic and induced pluripotent stem cells.

Dynamic regulation of miRNA expression in ordered stages of cellular development.

Short RNA expression in several distinct stages of T-lymphocyte development was comprehensively profiled. The total number of microRNAs (miRNAs) expressed per cell at different stages of development varies over nearly an order of magnitude in parallel with changes in total cellular RNA content, suggesting that global miRNA levels are coregulated with the translational capacity of the cell. However, individual miRNAs were dynamically regulated during T-cell development, with at least one miRNA or miRNA family overrepresented at each developmental stage.

Pegasys: software for executing and integrating analyses of biological sequences.

Background: We present Pegasys--a flexible, modular and customizable software system that facilitates the execution and data integration from heterogeneous biological sequence analysis tools.

Results: The Pegasys system includes numerous tools for pair-wise and multiple sequence alignment, ab initio gene prediction, RNA gene detection, masking repetitive sequences in genomic DNA as well as filters for database formatting and processing raw output from various analysis tools. We introduce a novel data structure for creating workflows of sequence analyses and a unified data model to store its results.