A transcribed, multi-channel, and continuously evolving molecular recorder 77 To achieve our goal of a tunable, high information content molecular recorder, we 78 utilized Cas9 to generate insertions or deletions (indels) upon repair of double-stranded breaks, 79 which are inherited in the next generation of cells 11-16. We record within a 205 base pair, synthetic DNA "target site" containing three "cut sites" and a static 8 base pair "integration barcode" (intBC), which are delivered in multiple copies via piggyBac transposition (Fig. 1a, b). We embedded this sequence into the 3'UTR of a constitutively transcribed fluorescent protein to enable profiling from the transcriptome. A second cassette encodes three independently transcribed and complementary guide RNAs to permit recording of multiple, distinct signals (Fig. 1a, b) 18. Our system is capable of high information storage due to the diversity of heritable repair outcomes, and the large number of targeted sites, which can be distinguished by the intBC (Fig. 1c). DNA repair generates hundreds of unique indels, and the distribution for each cut site is different and nonuniform: some produce highly biased outcomes while others create a diverse series (Fig. 1c, Extended Data Fig. 1) 19-21. To identify sequences that can tune the mutation rate of our recorder for timescales that are not pre-defined, and may extend from days to months, we screened several guide RNA series containing mismatches to their targets 22 by monitoring their activity on a GFP reporter over a 20-day timecourse and selected those that demonstrated a broad dynamic range (Fig. 1d). Slower cutting rates may improve viability in vivo, as frequent Cas9mediated double-strand breaks can cause cellular toxicity 23,24. To demonstrate information recovery from single cell transcriptomes, we stably transduced K562 cells with our technology and generated a primary, cell-barcoded cDNA pool via the 10x Genomics platform, allowing us * * *

SUMMARY To exert regulatory function, miRNAs guide Argonaute (AGO) proteins to partially complementary sites on target RNAs. Crosslinking and immunoprecipitation (“CLIP”) assays are state-of-the-art to map AGO binding sites, but assigning the targeting miRNA to these sites relies on bioinformatics predictions and is therefore indirect. To directly and unambiguously identify miRNA:target site interactions, we modified our CLIP methodology in C. elegans to experimentally ligate miRNAs to their target sites. Unexpectedly, ligation reactions also occurred in absence of the exogenous ligase. Our in vivo dataset and re-analysis of published mammalian AGO-CLIP data for miRNA-chimeras yielded ~17,000 miRNA:target site interactions. Analysis of interactions and extensive experimental validation of chimera-discovered targets of viral miRNAs suggest that our strategy identifies canonical, noncanonical, and nonconserved miRNA interactions. Our data suggest that ~80% of miRNA interactions have perfect or partial seed complementarity. In summary, analysis of miRNA:target chimeras enables the systematic, context-specific, in vivo discovery of miRNA binding.

BackgroundRecent developments in droplet-based microfluidics allow the transcriptional profiling of thousands of individual cells in a quantitative, highly parallel and cost-effective way. A critical, often limiting step is the preparation of cells in an unperturbed state, not altered by stress or ageing. Other challenges are rare cells that need to be collected over several days or samples prepared at different times or locations.MethodsHere, we used chemical fixation to address these problems. Methanol fixation allowed us to stabilise and preserve dissociated cells for weeks without compromising single-cell RNA sequencing data.ResultsBy using mixtures of fixed, cultured human and mouse cells, we first showed that individual transcriptomes could be confidently assigned to one of the two species. Single-cell gene expression from live and fixed samples correlated well with bulk mRNA-seq data. We then applied methanol fixation to transcriptionally profile primary cells from dissociated, complex tissues. Low RNA content cells from Drosophila embryos, as well as mouse hindbrain and cerebellum cells prepared by fluorescence-activated cell sorting, were successfully analysed after fixation, storage and single-cell droplet RNA-seq. We were able to identify diverse cell populations, including neuronal subtypes. As an additional resource, we provide 'dropbead', an R package for exploratory data analysis, visualization and filtering of Drop-seq data.ConclusionsWe expect that the availability of a simple cell fixation method will open up many new opportunities in diverse biological contexts to analyse transcriptional dynamics at single-cell resolution.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-017-0383-5) contains supplementary material, which is available to authorized users.

Summary paragraph: During ontogeny, proliferating cells become restricted in their fate through the combined action of cell-type specific transcription factors and ubiquitous epigenetic machinery, which recognize universally available histone residues or nucleotides but are nonetheless deployed in a highly context-dependent manner 1 , 2 . The molecular functions of these regulators are generally well understood, but assigning direct developmental roles is hampered by complex mutant phenotypes that often emerge following gastrulation 3 , 4 . Recently, single-cell RNA sequencing (scRNA-seq) and analytical approaches have explored this highly conserved process across numerous model organisms 5 – 8 , including mouse 9 – 18 . To elaborate on these strategies, we investigated a panel of ten essential regulators using a combined zygotic perturbation, scRNA-seq platform where many mutant embryos can be assayed simultaneously to recover robust transcriptional and morphological information. Deeper analysis of central Polycomb Repressive Complex (PRC) 1 and 2 members indicate substantial cooperativity, but distinguishes a PRC2-dominant role in restricting the germline that emerges from gross molecular changes within the initial conceptus. We believe our experimental framework will eventually allow for a fully quantitative view of how cellular diversity emerges using an identical genetic template and from a single totipotent cell.

Highlights d Repeat expansions in transcription factors (TFs) alter their phase separation capacity d Repeat expansions in TFs perturb the composition of TFcontaining condensates d Hoxd13 repeat expansions alter the transcriptional program in a synpolydactyly model d Features of intrinsically disordered regions in TFs are linked to condensation behavior

Background: Recent developments in droplet-based microfluidics allow the transcriptional profiling of

Post-transcriptional regulatory mechanisms are of fundamental importance to form robust genetic networks, but their roles in stem cell pluripotency remain poorly understood. Here, we use freshwater planarians as a model system to investigate this and uncover a role for CCR4-NOT mediated deadenylation of mRNAs in stem cell differentiation. Planarian adult stem cells, the so-called neoblasts, drive the almost unlimited regenerative capabilities of planarians and allow their ongoing homeostatic tissue turnover. While many genes have been demonstrated to be required for these processes, currently almost no mechanistic insight is available into their regulation. We show that knockdown of planarian Not1, the CCR4-NOT deadenylating complex scaffolding subunit, abrogates regeneration and normal homeostasis. This abrogation is primarily due to severe impairment of their differentiation potential. We describe a stem cell specific increase in the mRNA levels of key neoblast genes after Smed-not1 knock down, consistent with a role of the CCR4-NOT complex in degradation of neoblast mRNAs upon the onset of differentiation. We also observe a stem cell specific increase in the frequency of longer poly(A) tails in these same mRNAs, showing that stem cells after Smed-not1 knock down fail to differentiate as they accumulate populations of transcripts with longer poly(A) tails. As other transcripts are unaffected our data hint at a targeted regulation of these key stem cell mRNAs by post-transcriptional regulators such as RNA-binding proteins or microRNAs. Together, our results show that the CCR4-NOT complex is crucial for stem cell differentiation and controls stem cell-specific degradation of mRNAs, thus providing clear mechanistic insight into this aspect of neoblast biology.

Chan, Smith et al. 2018 Molecular recording of mammalian embryogenesis 2 Understanding the emergence of complex multicellular organisms from single totipotent cells, or ontogenesis, represents a foundational question in biology. The study of mammalian development is particularly challenging due to the difficulty of monitoring embryos in utero, the variability of progenitor field sizes, and the indeterminate relationship between the generation of uncommitted progenitors and their progression to subsequent stages. Here, we present a flexible, high information, multi-channel molecular recorder with a single cell (sc) readout and apply it as an evolving lineage tracer to define a mouse cell fate map from fertilization through gastrulation. By combining lineage information with scRNA-seq profiles, we recapitulate canonical developmental relationships between different tissue types and reveal an unexpected transcriptional convergence of endodermal cells from extra-embryonic and embryonic origins, illustrating how lineage information complements scRNA-seq to define cell types. Finally, we apply our cell fate map to estimate the number of embryonic progenitor cells and the degree of asymmetric partitioning within the pluripotent epiblast during specification. Our approach enables massively parallel, high-resolution recording of lineage and other information in mammalian systems to facilitate a quantitative framework for describing developmental processes.