Fluorescence microscopy is a key method in the life sciences. State of the art -omics methods combine fluorescence microscopy with complex protocols to visualize tens to thousands of features in each ...of millions of pixels across samples. These -omics methods require precise control of temperature, reagent application, and image acquisition parameters during iterative chemistry and imaging cycles conducted over the course of days or weeks. Automated execution of such methods enables robust and reproducible data generation. However, few commercial solutions exist for temperature controlled, fluidics coupled fluorescence imaging, and implementation of bespoke instrumentation requires specialized engineering expertise. Here we present PySeq2500, an open source Python code base and flow cell design that converts the Illumina HiSeq 2500 instrument, comprising an epifluorescence microscope with integrated fluidics, into an open platform for programmable applications without need for specialized engineering or software development expertise. Customizable PySeq2500 protocols enable experimental designs involving simultaneous 4-channel image acquisition, temperature control, reagent exchange, stable positioning, and sample integrity over extended experiments. To demonstrate accessible automation of complex, multi-day workflows, we use the PySeq2500 system for unattended execution of iterative indirect immunofluorescence imaging (4i). Our automated 4i method uses off-the-shelf antibodies over multiple cycles of staining, imaging, and antibody elution to build highly multiplexed maps of cell types and pathological features in mouse and postmortem human spinal cord sections. Given the widespread availability of HiSeq 2500 platforms and the simplicity of the modifications required to repurpose these systems, PySeq2500 enables non-specialists to develop and implement state of the art fluidics coupled imaging methods in a widely available benchtop system.
Peptide exchange technologies are essential for the generation of pMHC-multimer libraries used to probe diverse, polyclonal TCR repertoires in various settings. Here, using the molecular chaperone ...TAPBPR, we develop a robust method for the capture of stable, empty MHC-I molecules comprising murine H2 and human HLA alleles, which can be readily tetramerized and loaded with peptides of choice in a high-throughput manner. Alternatively, catalytic amounts of TAPBPR can be used to exchange placeholder peptides with high affinity peptides of interest. Using the same system, we describe high throughput assays to validate binding of multiple candidate peptides on empty MHC-I/TAPBPR complexes. Combined with tetramer-barcoding via a multi-modal cellular indexing technology, ECCITE-seq, our approach allows a combined analysis of TCR repertoires and other T cell transcription profiles together with their cognate antigen specificities in a single experiment. The new approach allows TCR/pMHC interactions to be interrogated easily at large scale.
Immune cell activation assays have been widely used for immune monitoring and for understanding disease mechanisms. However, these assays are typically limited in scope. A holistic study of ...circulating immune cell responses to different activators is lacking. Here we developed a cost-effective high-throughput multiplexed single-cell RNA-seq combined with epitope tagging (CITE-seq) to determine how classic activators of T cells (anti-CD3 coupled with anti-CD28) or monocytes (LPS) alter the cell composition and transcriptional profiles of peripheral blood mononuclear cells (PBMCs) from healthy human donors. Anti-CD3/CD28 treatment activated all classes of lymphocytes either directly (T cells) or indirectly (B and NK cells) but reduced monocyte numbers. Activated T and NK cells expressed senescence and effector molecules, whereas activated B cells transcriptionally resembled autoimmune disease- or age-associated B cells (e.g., CD11c, T-bet). In contrast, LPS specifically targeted monocytes and induced two main states: early activation characterized by the expression of chemoattractants and a later pro-inflammatory state characterized by expression of effector molecules. These data provide a foundation for future immune activation studies with single cell technologies (https://czi-pbmc-cite-seq.jax.org/).
Simultaneous measurement of surface proteins and gene expression within single cells using oligo-conjugated antibodies offers high-resolution snapshots of complex cell populations. Signal from ...oligo-conjugated antibodies is quantified by high-throughput sequencing and is highly scalable and sensitive. We investigated the response of oligo-conjugated antibodies towards four variables: concentration, staining volume, cell number at staining, and tissue. We find that staining with recommended antibody concentrations causes unnecessarily high background and amount of antibody used can be drastically reduced without loss of biological information. Reducing staining volume only affects antibodies targeting abundant epitopes used at low concentrations and is counteracted by reducing cell numbers. Adjusting concentrations increases signal, lowers background, and reduces costs. Background signal can account for a major fraction of total sequencing and is primarily derived from antibodies used at high concentrations. This study provides new insight into titration response and background of oligo-conjugated antibodies and offers concrete guidelines to improve such panels.
We analyzed the usage and consequences of alternative cleavage and polyadenylation (APA) in Drosophila melanogaster by using >1 billion reads of stranded mRNA-seq across a variety of dissected ...tissues. Beyond demonstrating that a majority of fly transcripts are subject to APA, we observed broad trends for 3′ untranslated region (UTR) shortening in the testis and lengthening in the central nervous system (CNS); the latter included hundreds of unannotated extensions ranging up to 18 kb. Extensive northern analyses validated the accumulation of full-length neural extended transcripts, and in situ hybridization indicated their spatial restriction to the CNS. Genes encoding RNA binding proteins (RBPs) and transcription factors were preferentially subject to 3′ UTR extensions. Motif analysis indicated enrichment of miRNA and RBP sites in the neural extensions, and their termini were enriched in canonical cis elements that promote cleavage and polyadenylation. Altogether, we reveal broad tissue-specific patterns of APA in Drosophila and transcripts with unprecedented 3′ UTR length in the nervous system.
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► >1 billion stranded RNA-seq reads generated across a panel of Drosophila tissues ► Broad trends of 3′ UTR shortening in testis and lengthening in CNS observed ► CNS 3′ UTR extensions subject 100 s of genes to tissue-specific posttranscriptional control ► CNS 3′ UTR extensions enriched for genes encoding nucleic-acid-binding proteins
Alternative cleavage and polyadenylation is a widespread mechanism that generates 3′ UTR diversity. Using stranded mRNA-seq in Drosophila, Lai and colleagues uncover global trends for 3′ UTR shortening in the testis and lengthening in the CNS. Their extensive experimental validation demonstrates the extraordinary length of many previously unannotated 3′ UTR extensions and their CNS-specific expression. Enrichment of miRNA target sites and RNA binding protein sites in these extended 3′ UTRs suggests important regulatory roles for these extensions in the CNS.
microRNAs (miRNAs) comprise an extensive class of post-transcriptional regulatory molecules in higher eukaryotes. Intensive research in Drosophila has revealed that miRNAs control myriad ...developmental and physiological processes. Interestingly, several of the best-studied miRNAs impact multiple biological processes, often by regulating distinct key target genes in each setting. Here we discuss the roles of some of these pleiotropic miRNAs, and their implications for studying and interpreting the roles of miRNAs in gene regulatory networks.
BackgroundFFPE tissues are the most commonly generated sample type in clinical settings. They provide valuable diagnostic information about disease etiology. However, many existing technologies for ...profiling gene expression at the RNA level are either incompatible with FFPE tissues due to formaldehyde crosslinking and RNA degradation or lack the ability to resolve expression patterns at single cell resolution.MethodsThe new and highly sensitive Chromium Single Cell Gene Expression Flex assay (Flex) from 10x Genomics uses a probe-based approach to profile the whole transcriptome in fixed samples, including FFPE tissues. The Flex assay enables single cell sequencing of FFPE tissues (scFFPE-Seq) with highly sensitive detection of whole transcriptome gene expression.ResultsTo demonstrate the robustness of the Flex assay, we separately dissociated FFPE sections from 38 human tissue blocks containing both healthy and diseased/cancer samples derived from a variety of tissues including Alzheimer’s brain, glioblastoma, breast, colon, heart, liver, lung, ovary, prostate, and skin. Data derived from these samples provided important biological insights including distinct cell clustering along with identification of representative cell types. Additionally, consistent data quality observed across different sections from the same FFPE block highlights the reproducibility and reliability of the assay.The ability to integrate Flex single cell data with Visium CytAssist spatial data from the same FFPE block through spot deconvolution allows for a more comprehensive understanding of biology. Each spot in Visium CytAssist data may include multiple cells. Using spot deconvolution methods that annotate scFFPE data as reference, the proportion of different cell types in each Visium spot can be determined to further refine cell heterogeneity for spatial visualization. After data integration for a colon cancer sample, we identified distinct tumor stroma with plasma cells expressing MZB1 surrounding tumor regions with high expression of BRCA1, nicely overlapping the H&E images. The integration of both platforms opens opportunities to leverage single cell resolution while preserving spatial context.ConclusionsIn summary, Chromium Single Cell Gene Expression Flex enables characterization of the biology preserved in human FFPE tumor samples at a single cell level. The assay expands the capabilities of 10x Genomics’ Chromium platform, enabling cross-assay compatibility with Visium CytAssist Spatial Gene Expression for FFPE samples and serves as a powerful tool to facilitate discoveries in disease progression and therapeutic target development.
Although Dicer is essential for general microRNA (miRNA) biogenesis, vertebrate mir-451 is Dicer independent. Instead, its short pre-miRNA hairpin is 'sliced' by Ago2, then 3'-resected into mature ...miRNAs. Here, we show that Drosophila cells and animals generate functional small RNAs from mir-451-type precursors. However, their bulk maturation arrests as Ago-cleaved pre-miRNAs, which mostly associate with the RNAi effector AGO2. Routing of pre-mir-451 hairpins to the miRNA effector AGO1 was inhibited by Dicer-1 and its partner Loqs. Loss of these miRNA factors promoted association of pre-mir-451 with AGO1, which sliced them and permitted maturation into ∼ 23-26 nt products. The difference was due to the 3' modification of single-stranded species in AGO2 by Hen1 methyltransferase, whose depletion permitted 3' trimming of Ago-cleaved pre-miRNAs in AGO2. Surprisingly, Nibbler, a 3'-5' exoribonuclease that trims 'long' mature miRNAs in AGO1, antagonized miR-451 processing. We used an in vitro reconstitution assay to identify a soluble, EDTA-sensitive activity that resects sliced pre-miRNAs in AGO1 complexes. Finally, we use deep sequencing to show that depletion of dicer-1 increases the diversity of small RNAs in AGO1, including some candidate mir-451-like loci. Altogether, we document unexpected aspects of miRNA biogenesis and Ago sorting, and provide insights into maturation of Argonaute-cleaved miRNA substrates.
Clinical outcomes in colorectal cancer (CRC) correlate with T cell infiltrates, but the specific contributions of heterogenous T cell types remain unclear. To investigate the diverse function of T ...cells in CRC, we profiled 37,931 T cells from tumors and adjacent normal colon of 16 patients with CRC with respect to transcriptome, TCR sequence, and cell surface markers. Our analysis identified phenotypically and functionally distinguishable effector T cell types. We employed single-cell gene signatures from these T cell subsets to query the TCGA database to assess their prognostic significance. We found 2 distinct cytotoxic T cell types. GZMK+KLRG1+ cytotoxic T cells were enriched in CRC patients with good outcomes. GNLY+CD103+ cytotoxic T cells with a dysfunctional phenotype were not associated with good outcomes, despite coexpression of CD39 and CD103, markers that denote tumor reactivity. We found 2 distinct Treg subtypes associated with opposite outcomes. While total Tregs were associated with good outcomes, CD38+ Tregs were associated with bad outcomes independently of stage and possessed a highly suppressive phenotype, suggesting that they inhibit antitumor immunity in CRC. These findings highlight the potential utility of these subpopulations in predicting outcomes and support the potential for novel therapies directed at CD38+ Tregs or CD8+CD103+ T cells.
It is apparent that microRNAs (miRNAs) are important components in the regulation of genetic networks in many biological contexts. Based on computational analysis, typical miRNAs are inferred to have ...tens to hundreds of conserved targets. Many miRNA-target interactions have been validated by various means, including heterologous tests in cultured cells and gain-of-function approaches that can yield striking phenotypes in whole animals. However, these strategies do not report on the endogenous importance of such miRNA activities. Likewise, studies of miRNA pathway mutants can suggest an endogenous role for miRNAs in a given setting, but do not identify roles for specific miRNAs. Therefore, these approaches must be complemented with the analysis of miRNA mutant alleles. In this review, we describe some of the lessons learned from studying miRNA gene deletions in worms, flies and mice, and discuss their implications for the control of endogenous regulatory networks.
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