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Vincent Croset, Christoph D Treiber, Scott Waddell
To understand the brain, molecular details need to be overlaid onto neural wiring diagrams so that synaptic mode, neuromodulation and critical signaling operations can be considered. Single-cell transcriptomics provide a unique opportunity to collect this information. Here we present an initial analysis of thousands of individual cells from Drosophila midbrain, that were acquired using Drop-Seq. A number of approaches permitted the assignment of transcriptional profiles to several major brain regions and cell-types...
April 19, 2018: ELife
Charles Cole, Ashley Byrne, Anna E Beaudin, E Camilla Forsberg, Christopher Vollmers
RNA-sequencing (RNA-seq) is a powerful technique to investigate and quantify entire transcriptomes. Recent advances in the field have made it possible to explore the transcriptomes of single cells. However, most widely used RNA-seq protocols fail to provide crucial information regarding transcription start sites. Here we present a protocol, Tn5Prime, that takes advantage of the Tn5 transposase-based Smart-seq2 protocol to create RNA-seq libraries that capture the 5' end of transcripts. The Tn5Prime method dramatically streamlines the 5' capture process and is both cost effective and reliable...
March 14, 2018: Nucleic Acids Research
Shiwei Zheng, Efthymia Papalexi, Andrew Butler, William Stephenson, Rahul Satija
Hematopoietic stem cells (HSCs) give rise to diverse cell types in the blood system, yet our molecular understanding of the early trajectories that generate this enormous diversity in humans remains incomplete. Here, we leverage Drop-seq, a massively parallel single-cell RNA sequencing (scRNA-seq) approach, to individually profile 20,000 progenitor cells from human cord blood, without prior enrichment or depletion for individual lineages based on surface markers. Our data reveal a transcriptional compendium of progenitor states in human cord blood, representing four committed lineages downstream from HSC, alongside the transcriptional dynamics underlying fate commitment...
March 15, 2018: Molecular Systems Biology
Frederique Ruf-Zamojski, Miguel Fribourg, Yongchao Ge, Venugopalan Nair, Hanna Pincas, Elena Zaslavsky, German Nudelman, Stephanie J Tuminello, Hideo Watanabe, Judith L Turgeon, Stuart C Sealfon
The LβT2 mouse pituitary cell line has many characteristics of a mature gonadotrope and is a widely used model system for studying the developmental processes and the response to gonadotropin-releasing hormone (GnRH). The global epigenetic landscape, which contributes to cell-specific gene regulatory mechanisms, and the single-cell transcriptome response variation of LβT2 cells have not been previously investigated. Here, we integrate the transcriptome and genome-wide chromatin accessibility state of LβT2 cells during GnRH stimulation...
2018: Frontiers in Endocrinology
Saket Jain, Darryl D Glubrecht, Devon R Germain, Markus Moser, Roseline Godbout
AP-2 transcription factors play important roles in the regulation of gene expression during development. Four of the five members of the AP-2 family (AP-2α, AP-2β, AP-2γ and AP-2δ) have previously been shown to be expressed in developing retina. Mouse knockouts have revealed roles for AP-2α, AP-2β and AP-2δ in retinal cell specification and function. Here, we show that the fifth member of the AP-2 family, AP-2ε, is also expressed in amacrine cells in developing mammalian and chicken retina. Our data indicate that there are considerably fewer AP-2ε-positive cells in the developing mouse retina compared to AP-2α, AP-2β and AP-2γ-positive cells, suggesting a specialized role for AP-2ε in a subset of amacrine cells...
February 21, 2018: Scientific Reports
Hui-Sung Moon, Kwanghwi Je, Jae-Woong Min, Donghyun Park, Kyung-Yeon Han, Seung-Ho Shin, Woong-Yang Park, Chang Eun Yoo, Shin-Hyun Kim
Single-cell RNA-seq reveals the cellular heterogeneity inherent in the population of cells, which is very important in many clinical and research applications. Recent advances in droplet microfluidics have achieved the automatic isolation, lysis, and labeling of single cells in droplet compartments without complex instrumentation. However, barcoding errors occurring in the cell encapsulation process because of the multiple-beads-in-droplet and insufficient throughput because of the low concentration of beads for avoiding multiple-beads-in-a-droplet remain important challenges for precise and efficient expression profiling of single cells...
February 27, 2018: Lab on a Chip
Bliss Magella, Mike Adam, Andrew S Potter, Meenakshi Venkatasubramanian, Kashish Chetal, Stuart B Hay, Nathan Salomonis, S Steven Potter
The developing kidney provides a useful model for study of the principles of organogenesis. In this report we use three independent platforms, Drop-Seq, Chromium 10x Genomics and Fluidigm C1, to carry out single cell RNA-Seq (scRNA-Seq) analysis of the E14.5 mouse kidney. Using the software AltAnalyze, in conjunction with the unsupervised approach ICGS, we were unable to identify and confirm the presence of 16 distinct cell populations during this stage of active nephrogenesis. Using a novel integrative supervised computational strategy, we were able to successfully harmonize and compare the cell profiles across all three technological platforms...
February 1, 2018: Developmental Biology
Kelvin Y Kwan
The spiral ganglion neurons (SGNs) of the cochlea are essential for our ability to hear. SGN loss after exposure to ototoxic drugs or loud noise results in hearing loss. Pluripotent stem cell-derived and endogenous progenitor cell types have the potential to become SGNs and are cellular foundations for replacement therapies. Repurposing transcriptional regulatory networks to promote SGN differentiation from progenitor cells is a strategy for regeneration. Advances in the Fludigm C1 workflow or Drop-seq allow sequencing of single cell transcriptomes to reveal variability between cells...
October 2016: Current Pharmacology Reports
Allon M Klein, Evan Macosko
No abstract text is available yet for this article.
July 25, 2017: Lab on a Chip
Jonathan Alles, Nikos Karaiskos, Samantha D Praktiknjo, Stefanie Grosswendt, Philipp Wahle, Pierre-Louis Ruffault, Salah Ayoub, Luisa Schreyer, Anastasiya Boltengagen, Carmen Birchmeier, Robert Zinzen, Christine Kocks, Nikolaus Rajewsky
BACKGROUND: Recent 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. METHODS: Here, we used chemical fixation to address these problems...
May 19, 2017: BMC Biology
Christoph Ziegenhain, Beate Vieth, Swati Parekh, Björn Reinius, Amy Guillaumet-Adkins, Martha Smets, Heinrich Leonhardt, Holger Heyn, Ines Hellmann, Wolfgang Enard
Single-cell RNA sequencing (scRNA-seq) offers new possibilities to address biological and medical questions. However, systematic comparisons of the performance of diverse scRNA-seq protocols are lacking. We generated data from 583 mouse embryonic stem cells to evaluate six prominent scRNA-seq methods: CEL-seq2, Drop-seq, MARS-seq, SCRB-seq, Smart-seq, and Smart-seq2. While Smart-seq2 detected the most genes per cell and across cells, CEL-seq2, Drop-seq, MARS-seq, and SCRB-seq quantified mRNA levels with less amplification noise due to the use of unique molecular identifiers (UMIs)...
February 16, 2017: Molecular Cell
John N Campbell, Evan Z Macosko, Henning Fenselau, Tune H Pers, Anna Lyubetskaya, Danielle Tenen, Melissa Goldman, Anne M J Verstegen, Jon M Resch, Steven A McCarroll, Evan D Rosen, Bradford B Lowell, Linus T Tsai
The hypothalamic arcuate-median eminence complex (Arc-ME) controls energy balance, fertility and growth through molecularly distinct cell types, many of which remain unknown. To catalog cell types in an unbiased way, we profiled gene expression in 20,921 individual cells in and around the adult mouse Arc-ME using Drop-seq. We identify 50 transcriptionally distinct Arc-ME cell populations, including a rare tanycyte population at the Arc-ME diffusion barrier, a new leptin-sensing neuron population, multiple agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) subtypes, and an orexigenic somatostatin neuron population...
March 2017: Nature Neuroscience
W Miachel Hanson, Zhe Chen, Laurie K Jackson, Meriem Attaf, Andrew K Sewell, Jennifer M Heemstra, John D Phillips
Next-generation sequencing (NGS) has proven to be an exceptionally powerful tool for studying genetic variation and differences in gene expression profiles between cell populations. However, these population-wide studies are limited by their inability to detect variation between individual cells within a population, inspiring the development of single-cell techniques such as Drop-seq, which add a unique barcode to the mRNA from each cell prior to sequencing. Current Drop-seq technology enables capture, amplification, and barcoding of the entire mRNA transcriptome of individual cells...
September 7, 2016: Journal of the American Chemical Society
Xiao Huo, Sheng'en Hu, Chengchen Zhao, Yong Zhang
MOTIVATION: Drop-seq has recently emerged as a powerful technology to analyze gene expression from thousands of individual cells simultaneously. Currently, Drop-seq technology requires refinement and quality control (QC) steps are critical for such data analysis. There is a strong need for a convenient and comprehensive approach to obtain dedicated QC and to determine the relationships between cells for ultra-high-dimensional datasets. RESULTS: We developed Dr.seq, a QC and analysis pipeline for Drop-seq data...
July 15, 2016: Bioinformatics
Anthony Hickey, Caroline Esnault, Anasuya Majumdar, Atreyi Ghatak Chatterjee, James R Iben, Philip G McQueen, Andrew X Yang, Takeshi Mizuguchi, Shiv I S Grewal, Henry L Levin
Transposable elements (TEs) constitute a substantial fraction of the eukaryotic genome and, as a result, have a complex relationship with their host that is both adversarial and dependent. To minimize damage to cellular genes, TEs possess mechanisms that target integration to sequences of low importance. However, the retrotransposon Tf1 of Schizosaccharomyces pombe integrates with a surprising bias for promoter sequences of stress-response genes. The clustering of integration in specific promoters suggests that Tf1 possesses a targeting mechanism that is important for evolutionary adaptation to changes in environment...
November 2015: Genetics
Hongzheng Sun, Ting Peng, Yafan Zhao, Yanxiu Du, Jing Zhang, Junzhou Li, Zeyu Xin, Quanzhi Zhao
Poor grain filling of inferior grains located on lower secondary panicle branch causes great drop in rice yield and quality. Dynamic gene expression patterns between superior and inferior grains were examined from the view of the whole transcriptome by using RNA-Seq method. In total, 19,442 genes were detected during rice grain development. Genes involved in starch synthesis, grain storage and grain development were interrogated in particular in superior and inferior grains. Of the genes involved in sucrose to starch transformation process, most were expressed at lower level in inferior grains at early filling stage compared to that of superior grains...
2015: PloS One
Jae-Woong Min, Woo Jin Kim, Jeong A Han, Yu-Jin Jung, Kyu-Tae Kim, Woong-Yang Park, Hae-Ock Lee, Sun Shim Choi
Single-cell sequencing, which is used to detect clinically important tumor subpopulations, is necessary for understanding tumor heterogeneity. Here, we analyzed transcriptomic data obtained from 34 single cells from human lung adenocarcinoma (LADC) patient-derived xenografts (PDXs). To focus on the intrinsic transcriptomic signatures of these tumors, we filtered out genes that displayed extensive expression changes following xenografting and cell culture. Then, we performed clustering analysis using co-regulated gene modules rather than individual genes to minimize read drop-out errors associated with single-cell sequencing...
2015: PloS One
Zixia Huang, Aurore Gallot, Nga T Lao, Sébastien J Puechmaille, Nicole M Foley, David Jebb, Michaël Bekaert, Emma C Teeling
The acquisition of tissue samples from wild populations is a constant challenge in conservation biology, especially for endangered species and protected species where nonlethal sampling is the only option. Whole blood has been suggested as a nonlethal sample type that contains a high percentage of bodywide and genomewide transcripts and therefore can be used to assess the transcriptional status of an individual, and to infer a high percentage of the genome. However, only limited quantities of blood can be nonlethally sampled from small species and it is not known if enough genetic material is contained in only a few drops of blood, which represents the upper limit of sample collection for some small species...
January 2016: Molecular Ecology Resources
Assaf Rotem, Oren Ram, Noam Shoresh, Ralph A Sperling, Michael Schnall-Levin, Huidan Zhang, Anindita Basu, Bradley E Bernstein, David A Weitz
The importance of single-cell level data is increasingly appreciated, and significant advances in this direction have been made in recent years. Common to these technologies is the need to physically segregate individual cells into containers, such as wells or chambers of a micro-fluidics chip. High-throughput Single-Cell Labeling (Hi-SCL) in drops is a novel method that uses drop-based libraries of oligonucleotide barcodes to index individual cells in a population. The use of drops as containers, and a microfluidics platform to manipulate them en-masse, yields a highly scalable methodological framework...
2015: PloS One
Evan Z Macosko, Anindita Basu, Rahul Satija, James Nemesh, Karthik Shekhar, Melissa Goldman, Itay Tirosh, Allison R Bialas, Nolan Kamitaki, Emily M Martersteck, John J Trombetta, David A Weitz, Joshua R Sanes, Alex K Shalek, Aviv Regev, Steven A McCarroll
Cells, the basic units of biological structure and function, vary broadly in type and state. Single-cell genomics can characterize cell identity and function, but limitations of ease and scale have prevented its broad application. Here we describe Drop-seq, a strategy for quickly profiling thousands of individual cells by separating them into nanoliter-sized aqueous droplets, associating a different barcode with each cell's RNAs, and sequencing them all together. Drop-seq analyzes mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts' cell of origin...
May 21, 2015: Cell
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