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single cell RNA sequencing,scRNA-seq

Haojia Wu, Yuhei Kirita, Erinn L Donnelly, Benjamin D Humphreys
BACKGROUND: A challenge for single-cell genomic studies in kidney and other solid tissues is generating a high-quality single-cell suspension that contains rare or difficult-to-dissociate cell types and is free of both RNA degradation and artifactual transcriptional stress responses. METHODS: We compared single-cell RNA sequencing (scRNA-seq) using the DropSeq platform with single-nucleus RNA sequencing (snRNA-seq) using sNuc-DropSeq, DroNc-seq, and 10X Chromium platforms on adult mouse kidney...
December 3, 2018: Journal of the American Society of Nephrology: JASN
Kent A Riemondy, Monica Ransom, Christopher Alderman, Austin E Gillen, Rui Fu, Jessica Finlay-Schultz, Gregory D Kirkpatrick, Jorge Di Paola, Peter Kabos, Carol A Sartorius, Jay R Hesselberth
Single-cell RNA sequencing (scRNA-seq) methods generate sparse gene expression profiles for thousands of single cells in a single experiment. The information in these profiles is sufficient to classify cell types by distinct expression patterns but the high complexity of scRNA-seq libraries often prevents full characterization of transcriptomes from individual cells. To extract more focused gene expression information from scRNA-seq libraries, we developed a strategy to physically recover the DNA molecules comprising transcriptome subsets, enabling deeper interrogation of the isolated molecules by another round of DNA sequencing...
November 29, 2018: Nucleic Acids Research
Angela Barrett, Chui-Yee Fong, Arjuna Subramanian, Wenting Liu, Yirui Feng, Mahesh Choolani, Arijit Biswas, Jagath Rajapakse, Ariff Bongso
Human Wharton's jelly stem cells (hWJSCs) isolated from the human umbilical cord are a unique population of mesenchymal stem cells with significant clinical utility. Their broad differentiation potential, high rate of proliferation, ready availability from discarded cords, and prolonged maintenance of stemness properties in culture make them an attractive alternative source of MSCs with therapeutic value compared to bone marrow MSCs (hBMMSCs). We aimed to characterise the differences in gene expression profiles between these two stem cell types using single-cell RNA sequencing (scRNA-Seq) to determine which pathways are involved in conferring hWJSCs with their unique properties...
November 28, 2018: Stem Cells and Development
Majd M Ariss, Abul B M M K Islam, Meg Critcher, Maria Paula Zappia, Maxim V Frolov
The function of Retinoblastoma tumor suppressor (pRB) is greatly influenced by the cellular context, therefore the consequences of pRB inactivation are cell-type-specific. Here we employ single cell RNA-sequencing (scRNA-seq) to profile the impact of an Rbf mutation during Drosophila eye development. First, we build a catalogue of 11,500 wild type eye disc cells containing major known cell types. We find a transcriptional switch occurring in differentiating photoreceptors at the time of axonogenesis. Next, we map a cell landscape of Rbf mutant and identify a mutant-specific cell population that shows intracellular acidification due to increase in glycolytic activity...
November 27, 2018: Nature Communications
Yong Yu, Pengtao Liu
Single-cell RNA sequencing (scRNA-seq) is a powerful tool to study immune cells, which enables an unbiased way to discover novel cell populations, biological meaningful cellular heterogeneity, and cell lineage development trajectories. Advances in scRNA-seq technologies and computational data analysis have driven a revolution in our understanding of the immune system in health and disease. Technically, the key step for scRNA-seq analysis is making a high-quality cDNA library for sequencing. Here, we describe a plate-based protocol to prepare single-cell cDNA library of bone marrow innate lymphoid precursors for next generation sequencing-based transcriptome analysis...
2019: Methods in Molecular Biology
Burak Dura, Jin-Young Choi, Kerou Zhang, William Damsky, Durga Thakral, Marcus Bosenberg, Joe Craft, Rong Fan
Cellular barcoding of 3' mRNAs enabled massively parallel profiling of single-cell gene expression and has been implemented in droplet and microwell based platforms. The latter further adds the value for compatibility with low input samples, optical imaging, scalability, and portability. However, cell lysis in microwells remains challenging despite the recently developed sophisticated solutions. Here, we present scFTD-seq, a microchip platform for performing single-cell freeze-thaw lysis directly toward 3' mRNA sequencing...
November 20, 2018: Nucleic Acids Research
Donghyung Lee, Anthony Cheng, Nathan Lawlor, Mohan Bolisetty, Duygu Ucar
Single cell RNA-sequencing (scRNA-seq) precisely characterizes gene expression levels and dissects variation in expression associated with the state (technical or biological) and the type of the cell, which is averaged out in bulk measurements. Multiple and correlated sources contribute to gene expression variation in single cells, which makes their estimation difficult with the existing methods developed for batch correction (e.g., surrogate variable analysis (SVA)) that estimate orthogonal transformations of these sources...
November 19, 2018: Scientific Reports
Bob Chen, Charles A Herring, Ken S Lau
Motivation: The emergence of single-cell RNA-sequencing (scRNA-seq) has enabled analyses that leverage transitioning cell states to reconstruct pseudotemporal trajectories. Multidimensional data sparsity, zero inflation, and technical variation necessitate the selection of high-quality features that feed downstream analyses. Despite the development of numerous algorithms for the unsupervised selection of biologically relevant features, their differential performance remains largely unaddressed...
November 16, 2018: Bioinformatics
Sumit Mukherjee, Alberto Carignano, Georg Seelig, Su-In Lee
Identifying the gene regulatory networks that control development or disease is one of the most important problems in biology. Here, we introduce a computational approach, called PIPER (ProgressIve network PERturbation), to identify the perturbed genes that drive differences in the gene regulatory network across different points in a biological progression. PIPER employs algorithms tailor-made for single cell RNA sequencing (scRNA-seq) data to jointly identify gene networks for multiple progressive conditions...
July 2018: Conference Proceedings: Annual International Conference of the IEEE Engineering in Medicine and Biology Society
Divyanshu Talwar, Aanchal Mongia, Debarka Sengupta, Angshul Majumdar
The emergence of single-cell RNA sequencing (scRNA-seq) technologies has enabled us to measure the expression levels of thousands of genes at single-cell resolution. However, insufficient quantities of starting RNA in the individual cells cause significant dropout events, introducing a large number of zero counts in the expression matrix. To circumvent this, we developed an autoencoder-based sparse gene expression matrix imputation method. AutoImpute, which learns the inherent distribution of the input scRNA-seq data and imputes the missing values accordingly with minimal modification to the biologically silent genes...
November 5, 2018: Scientific Reports
Livnat Jerby-Arnon, Parin Shah, Michael S Cuoco, Christopher Rodman, Mei-Ju Su, Johannes C Melms, Rachel Leeson, Abhay Kanodia, Shaolin Mei, Jia-Ren Lin, Shu Wang, Bokang Rabasha, David Liu, Gao Zhang, Claire Margolais, Orr Ashenberg, Patrick A Ott, Elizabeth I Buchbinder, Rizwan Haq, F Stephen Hodi, Genevieve M Boland, Ryan J Sullivan, Dennie T Frederick, Benchun Miao, Tabea Moll, Keith T Flaherty, Meenhard Herlyn, Russell W Jenkins, Rohit Thummalapalli, Monika S Kowalczyk, Israel Cañadas, Bastian Schilling, Adam N R Cartwright, Adrienne M Luoma, Shruti Malu, Patrick Hwu, Chantale Bernatchez, Marie-Andrée Forget, David A Barbie, Alex K Shalek, Itay Tirosh, Peter K Sorger, Kai Wucherpfennig, Eliezer M Van Allen, Dirk Schadendorf, Bruce E Johnson, Asaf Rotem, Orit Rozenblatt-Rosen, Levi A Garraway, Charles H Yoon, Benjamin Izar, Aviv Regev
Immune checkpoint inhibitors (ICIs) produce durable responses in some melanoma patients, but many patients derive no clinical benefit, and the molecular underpinnings of such resistance remain elusive. Here, we leveraged single-cell RNA sequencing (scRNA-seq) from 33 melanoma tumors and computational analyses to interrogate malignant cell states that promote immune evasion. We identified a resistance program expressed by malignant cells that is associated with T cell exclusion and immune evasion. The program is expressed prior to immunotherapy, characterizes cold niches in situ, and predicts clinical responses to anti-PD-1 therapy in an independent cohort of 112 melanoma patients...
November 1, 2018: Cell
Marmar Moussa, Ion I Măndoiu
BACKGROUND: Single cell transcriptomics is critical for understanding cellular heterogeneity and identification of novel cell types. Leveraging the recent advances in single cell RNA sequencing (scRNA-Seq) technology requires novel unsupervised clustering algorithms that are robust to high levels of technical and biological noise and scale to datasets of millions of cells. RESULTS: We present novel computational approaches for clustering scRNA-seq data based on the Term Frequency - Inverse Document Frequency (TF-IDF) transformation that has been successfully used in the field of text analysis...
August 13, 2018: BMC Genomics
Bushra Raj, James A Gagnon, Alexander F Schier
Lineage relationships among the large number of heterogeneous cell types generated during development are difficult to reconstruct in a high-throughput manner. We recently established a method, scGESTALT, that combines cumulative editing of a lineage barcode array by CRISPR-Cas9 with large-scale transcriptional profiling using droplet-based single-cell RNA sequencing (scRNA-seq). The technique generates edits in the barcode array over multiple timepoints using Cas9 and pools of single-guide RNAs (sgRNAs) introduced during early and late zebrafish embryonic development, which distinguishes it from similar Cas9 lineage-tracing methods...
November 2018: Nature Protocols
Qi Liu, Charles A Herring, Quanhu Sheng, Jie Ping, Alan J Simmons, Bob Chen, Amrita Banerjee, Wei Li, Guoqiang Gu, Robert J Coffey, Yu Shyr, Ken S Lau
Single-cell RNA sequencing (scRNA-seq) has become a powerful tool for the systematic investigation of cellular diversity. As a number of computational tools have been developed to identify and visualize cell populations within a single scRNA-seq dataset, there is a need for methods to quantitatively and statistically define proportional shifts in cell population structures across datasets, such as expansion or shrinkage or emergence or disappearance of cell populations. Here we present sc-UniFrac, a framework to statistically quantify compositional diversity in cell populations between single-cell transcriptome landscapes...
October 2018: PLoS Biology
Sokratis A Apostolidis, Giuseppina Stifano, Tracy Tabib, Lisa M Rice, Christina M Morse, Bashar Kahaleh, Robert Lafyatis
Objective: The mechanisms that lead to endothelial cell (EC) injury and propagate the vasculopathy in Systemic Sclerosis (SSc) are not well understood. Using single cell RNA sequencing (scRNA-seq), our goal was to identify EC markers and signature pathways associated with vascular injury in SSc skin. Methods: We implemented single cell sorting and subsequent RNA sequencing of cells isolated from SSc and healthy control skin. We used t-distributed stochastic neighbor embedding (t-SNE) to identify the various cell types...
2018: Frontiers in Immunology
Yuval Lieberman, Lior Rokach, Tal Shay
Single-cell RNA sequencing (scRNA-seq) is an emerging technology for profiling the gene expression of thousands of cells at the single cell resolution. Currently, the labeling of cells in an scRNA-seq dataset is performed by manually characterizing clusters of cells or by fluorescence-activated cell sorting (FACS). Both methods have inherent drawbacks: The first depends on the clustering algorithm used and the knowledge and arbitrary decisions of the annotator, and the second involves an experimental step in addition to the sequencing and cannot be incorporated into the higher throughput scRNA-seq methods...
2018: PloS One
Eoghainín Ó hAinmhire, Haojia Wu, Yoshiharu Muto, Erinn L Donnelly, Flavia G Machado, Lucy X Fan, Monica Chang-Panesso, Benjamin D Humphreys
Gli1-positive resident mesenchymal stem cell-like cells are the predominant source of kidney myofibroblasts in fibrosis but investigating Gli1-positive myofibroblast progenitor activation is hampered by the difficulty of isolating and propagating primary cultures of these cells. Using a genetic strategy with positive and negative selection, we isolated Kidney-Gli1 (KG1) cells that maintain expression of appropriate mesenchymal stem cell-like cell markers, respond to hedgehog pathway activation and display robust myofibroblast differentiation upon treatment with TGFb...
October 10, 2018: American Journal of Physiology. Renal Physiology
Denis Avey, Sumithra Sankararaman, Aldrin K Y Yim, Ruteja Barve, Jeffrey Milbrandt, Robi D Mitra
Molecular and behavioral responses to opioids are thought to be primarily mediated by neurons, although there is accumulating evidence that other cell types play a prominent role in drug addiction. To investigate cell-type-specific opioid responses, we performed single-cell RNA sequencing (scRNA-seq) of the nucleus accumbens of mice following acute morphine treatment. Differential expression analysis uncovered unique morphine-dependent transcriptional responses by oligodendrocytes and astrocytes. We examined the expression of selected genes, including Cdkn1a and Sgk1, by FISH, confirming their induction by morphine in oligodendrocytes...
September 25, 2018: Cell Reports
Eun-Ah Christine Song, Sangwon Min, Akinsola Oyelakin, Kirsten Smalley, Jonathan E Bard, Lan Liao, Jianming Xu, Rose-Anne Romano
Stem and progenitor cells of the submandibular salivary gland (SMG) give rise to, maintain, and regenerate the multiple lineages of mature epithelial cells including those belonging to the ductal, acinar, basal and myoepithelial subtypes. Here we have exploited single cell RNA-sequencing and in vivo genetic lineage tracing technologies to generate a detailed map of the cell fate trajectories and branch points of the basal and myoepithelial cell populations of the mouse SMG during embryonic development and in adults...
September 19, 2018: Scientific Reports
Shelli F Farhadian, Sameet S Mehta, Chrysoula Zografou, Kevin Robertson, Richard W Price, Jenna Pappalardo, Jennifer Chiarella, David A Hafler, Serena S Spudich
Central nervous system (CNS) immune activation is an important driver of neuronal injury during several neurodegenerative and neuroinflammatory diseases. During HIV infection, CNS immune activation is associated with high rates of neurocognitive impairment, even during sustained long-term suppressive antiretroviral therapy (ART). However, the cellular subsets that drive immune activation and neuronal damage in the CNS during HIV infection and other neurological conditions remain unknown, in part because CNS cells are difficult to access in living humans...
September 20, 2018: JCI Insight
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