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single nuclei RNA-seq

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https://www.readbyqxmd.com/read/30371670/isolation-of-adult-spinal-cord-nuclei-for-massively-parallel-single-nucleus-rna-sequencing
#1
Kaya J E Matson, Anupama Sathyamurthy, Kory R Johnson, Michael C Kelly, Matthew W Kelley, Ariel J Levine
Probing an individual cell's gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material...
October 12, 2018: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/30254108/single-nucleus-transcriptomic-survey-of-cell-diversity-and-functional-maturation-in-postnatal-mammalian-hearts
#2
Peng Hu, Jian Liu, Juanjuan Zhao, Benjamin J Wilkins, Katherine Lupino, Hao Wu, Liming Pei
A fundamental challenge in understanding cardiac biology and disease is that the remarkable heterogeneity in cell type composition and functional states have not been well characterized at single-cell resolution in maturing and diseased mammalian hearts. Massively parallel single-nucleus RNA sequencing (snRNA-seq) has emerged as a powerful tool to address these questions by interrogating the transcriptome of tens of thousands of nuclei isolated from fresh or frozen tissues. snRNA-seq overcomes the technical challenge of isolating intact single cells from complex tissues, including the maturing mammalian hearts; reduces biased recovery of easily dissociated cell types; and minimizes aberrant gene expression during the whole-cell dissociation...
October 1, 2018: Genes & Development
https://www.readbyqxmd.com/read/30143541/-pitx2-maintains-mitochondrial-function-during-regeneration-to-prevent-myocardial-fat-deposition
#3
Lele Li, Ge Tao, Matthew C Hill, Min Zhang, Yuka Morikawa, James F Martin
Loss of the paired-like homeodomain transcription factor 2 ( Pitx2 ) in cardiomyocytes predisposes mice to atrial fibrillation and compromises neonatal regenerative capacity. In addition, Pitx2 gain-of-function protects mature cardiomyocytes from ischemic injury and promotes heart repair. Here, we characterized the long-term myocardial phenotype following myocardial infarction (MI) in Pitx2 conditional-knockout ( Pitx2 CKO) mice. We found adipose-like tissue in Pitx2 CKO hearts 60 days after MI induced surgically at postnatal day 2 but not at day 8...
September 26, 2018: Development
https://www.readbyqxmd.com/read/30031009/single-sample-sequencing-s3eq-of-epigenome-and-transcriptome-in-nucleus-accumbens
#4
S J Xu, E A Heller
BACKGROUND: High-throughput sequencing has been widely applied to uncover the molecular mechanisms underlying neurological and psychiatric disorders. The large body of data support the role of epigenetic mechanisms in neurological function of both human and animals. Yet, the existing data is limited by the fact that epigenetic and transcriptomic changes have only been measured in separate cohorts. This has limited precise correlation of epigenetic changes in gene expression. NEW METHOD: Single Sample Sequencing (S3EQ) is an innovative approach to analyze both epigenetic and transcriptomic regulation within a single neuronal sample...
July 18, 2018: Journal of Neuroscience Methods
https://www.readbyqxmd.com/read/29545511/single-cell-profiling-of-the-developing-mouse-brain-and-spinal-cord-with-split-pool-barcoding
#5
Alexander B Rosenberg, Charles M Roco, Richard A Muscat, Anna Kuchina, Paul Sample, Zizhen Yao, Lucas T Graybuck, David J Peeler, Sumit Mukherjee, Wei Chen, Suzie H Pun, Drew L Sellers, Bosiljka Tasic, Georg Seelig
To facilitate scalable profiling of single cells, we developed split-pool ligation-based transcriptome sequencing (SPLiT-seq), a single-cell RNA-seq (scRNA-seq) method that labels the cellular origin of RNA through combinatorial barcoding. SPLiT-seq is compatible with fixed cells or nuclei, allows efficient sample multiplexing, and requires no customized equipment. We used SPLiT-seq to analyze 156,049 single-nucleus transcriptomes from postnatal day 2 and 11 mouse brains and spinal cords. More than 100 cell types were identified, with gene expression patterns corresponding to cellular function, regional specificity, and stage of differentiation...
April 13, 2018: Science
https://www.readbyqxmd.com/read/29321653/integrated-sequencing-of-exome-and-mrna-of-large-sized-single-cells
#6
Lily Yan Wang, Jiajie Guo, Wei Cao, Meng Zhang, Jiankui He, Zhoufang Li
Current approaches of single cell DNA-RNA integrated sequencing are difficult to call SNPs, because a large amount of DNA and RNA is lost during DNA-RNA separation. Here, we performed simultaneous single-cell exome and transcriptome sequencing on individual mouse oocytes. Using microinjection, we kept the nuclei intact to avoid DNA loss, while retaining the cytoplasm inside the cell membrane, to maximize the amount of DNA and RNA captured from the single cell. We then conducted exome-sequencing on the isolated nuclei and mRNA-sequencing on the enucleated cytoplasm...
January 10, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29220646/dissecting-cell-type-composition-and-activity-dependent-transcriptional-state-in-mammalian-brains-by-massively-parallel-single-nucleus-rna-seq
#7
Peng Hu, Emily Fabyanic, Deborah Y Kwon, Sheng Tang, Zhaolan Zhou, Hao Wu
Massively parallel single-cell RNA sequencing can precisely resolve cellular diversity in a high-throughput manner at low cost, but unbiased isolation of intact single cells from complex tissues such as adult mammalian brains is challenging. Here, we integrate sucrose-gradient-assisted purification of nuclei with droplet microfluidics to develop a highly scalable single-nucleus RNA-seq approach (sNucDrop-seq), which is free of enzymatic dissociation and nucleus sorting. By profiling ∼18,000 nuclei isolated from cortical tissues of adult mice, we demonstrate that sNucDrop-seq not only accurately reveals neuronal and non-neuronal subtype composition with high sensitivity but also enables in-depth analysis of transient transcriptional states driven by neuronal activity, at single-cell resolution, in vivo...
December 7, 2017: Molecular Cell
https://www.readbyqxmd.com/read/29180631/strt-seq-2i-dual-index-5-single-cell-and-nucleus-rna-seq-on-an-addressable-microwell-array
#8
Hannah Hochgerner, Peter Lönnerberg, Rebecca Hodge, Jaromir Mikes, Abeer Heskol, Hermann Hubschle, Philip Lin, Simone Picelli, Gioele La Manno, Michael Ratz, Jude Dunne, Syed Husain, Ed Lein, Maithreyan Srinivasan, Amit Zeisel, Sten Linnarsson
Single-cell RNA-seq has become routine for discovering cell types and revealing cellular diversity, but archived human brain samples still pose a challenge to current high-throughput platforms. We present STRT-seq-2i, an addressable 9600-microwell array platform, combining sampling by limiting dilution or FACS, with imaging and high throughput at competitive cost. We applied the platform to fresh single mouse cortical cells and to frozen post-mortem human cortical nuclei, matching the performance of a previous lower-throughput platform while retaining a high degree of flexibility, potentially also for other high-throughput applications...
November 27, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28846088/massively-parallel-single-nucleus-rna-seq-with-dronc-seq
#9
Naomi Habib, Inbal Avraham-Davidi, Anindita Basu, Tyler Burks, Karthik Shekhar, Matan Hofree, Sourav R Choudhury, François Aguet, Ellen Gelfand, Kristin Ardlie, David A Weitz, Orit Rozenblatt-Rosen, Feng Zhang, Aviv Regev
Single-nucleus RNA sequencing (sNuc-seq) profiles RNA from tissues that are preserved or cannot be dissociated, but it does not provide high throughput. Here, we develop DroNc-seq: massively parallel sNuc-seq with droplet technology. We profile 39,111 nuclei from mouse and human archived brain samples to demonstrate sensitive, efficient, and unbiased classification of cell types, paving the way for systematic charting of cell atlases.
October 2017: Nature Methods
https://www.readbyqxmd.com/read/28818938/comprehensive-single-cell-transcriptional-profiling-of-a-multicellular-organism
#10
Junyue Cao, Jonathan S Packer, Vijay Ramani, Darren A Cusanovich, Chau Huynh, Riza Daza, Xiaojie Qiu, Choli Lee, Scott N Furlan, Frank J Steemers, Andrew Adey, Robert H Waterston, Cole Trapnell, Jay Shendure
To resolve cellular heterogeneity, we developed a combinatorial indexing strategy to profile the transcriptomes of single cells or nuclei, termed sci-RNA-seq (single-cell combinatorial indexing RNA sequencing). We applied sci-RNA-seq to profile nearly 50,000 cells from the nematode Caenorhabditis elegans at the L2 larval stage, which provided >50-fold "shotgun" cellular coverage of its somatic cell composition. From these data, we defined consensus expression profiles for 27 cell types and recovered rare neuronal cell types corresponding to as few as one or two cells in the L2 worm...
August 18, 2017: Science
https://www.readbyqxmd.com/read/27897007/production-of-a-preliminary-quality-control-pipeline-for-single-nuclei-rna-seq-and-its-application-in-the-analysis-of-cell-type-diversity-of-post-mortem-human-brain-neocortex
#11
Brian Aevermann, Jamison McCorrison, Pratap Venepally, Rebecca Hodge, Trygve Bakken, Jeremy Miller, Mark Novotny, Danny N Tran, Francisco Diezfuertes, Lena Christiansen, Fan Zhang, Frank Steemers, Roger S Lasken, E D Lein, Nicholas Schork, Richard H Scheuermann
Next generation sequencing of the RNA content of single cells or single nuclei (sc/nRNA-seq) has become a powerful approach to understand the cellular complexity and diversity of multicellular organisms and environmental ecosystems. However, the fact that the procedure begins with a relatively small amount of starting material, thereby pushing the limits of the laboratory procedures required, dictates that careful approaches for sample quality control (QC) are essential to reduce the impact of technical noise and sample bias in downstream analysis applications...
2017: Pacific Symposium on Biocomputing
https://www.readbyqxmd.com/read/27566152/single-nucleus-rna-seq-of-differentiating-human-myoblasts-reveals-the-extent-of-fate-heterogeneity
#12
Weihua Zeng, Shan Jiang, Xiangduo Kong, Nicole El-Ali, Alexander R Ball, Christopher I-Hsing Ma, Naohiro Hashimoto, Kyoko Yokomori, Ali Mortazavi
Myoblasts are precursor skeletal muscle cells that differentiate into fused, multinucleated myotubes. Current single-cell microfluidic methods are not optimized for capturing very large, multinucleated cells such as myotubes. To circumvent the problem, we performed single-nucleus transcriptome analysis. Using immortalized human myoblasts, we performed RNA-seq analysis of single cells (scRNA-seq) and single nuclei (snRNA-seq) and found them comparable, with a distinct enrichment for long non-coding RNAs (lncRNAs) in snRNA-seq...
December 1, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27090946/nuclear-rna-seq-of-single-neurons-reveals-molecular-signatures-of-activation
#13
Benjamin Lacar, Sara B Linker, Baptiste N Jaeger, Suguna R Krishnaswami, Jerika J Barron, Martijn J E Kelder, Sarah L Parylak, Apuã C M Paquola, Pratap Venepally, Mark Novotny, Carolyn O'Connor, Conor Fitzpatrick, Jennifer A Erwin, Jonathan Y Hsu, David Husband, Michael J McConnell, Roger Lasken, Fred H Gage
Single-cell sequencing methods have emerged as powerful tools for identification of heterogeneous cell types within defined brain regions. Application of single-cell techniques to study the transcriptome of activated neurons can offer insight into molecular dynamics associated with differential neuronal responses to a given experience. Through evaluation of common whole-cell and single-nuclei RNA-sequencing (snRNA-seq) methods, here we show that snRNA-seq faithfully recapitulates transcriptional patterns associated with experience-driven induction of activity, including immediate early genes (IEGs) such as Fos, Arc and Egr1...
April 19, 2016: Nature Communications
https://www.readbyqxmd.com/read/26890679/using-single-nuclei-for-rna-seq-to-capture-the-transcriptome-of-postmortem-neurons
#14
Suguna Rani Krishnaswami, Rashel V Grindberg, Mark Novotny, Pratap Venepally, Benjamin Lacar, Kunal Bhutani, Sara B Linker, Son Pham, Jennifer A Erwin, Jeremy A Miller, Rebecca Hodge, James K McCarthy, Martin Kelder, Jamison McCorrison, Brian D Aevermann, Francisco Diez Fuertes, Richard H Scheuermann, Jun Lee, Ed S Lein, Nicholas Schork, Michael J McConnell, Fred H Gage, Roger S Lasken
A protocol is described for sequencing the transcriptome of a cell nucleus. Nuclei are isolated from specimens and sorted by FACS, cDNA libraries are constructed and RNA-seq is performed, followed by data analysis. Some steps follow published methods (Smart-seq2 for cDNA synthesis and Nextera XT barcoded library preparation) and are not described in detail here. Previous single-cell approaches for RNA-seq from tissues include cell dissociation using protease treatment at 30 °C, which is known to alter the transcriptome...
March 2016: Nature Protocols
https://www.readbyqxmd.com/read/25888089/rna-interference-screen-reveals-a-high-proportion-of-mitochondrial-proteins-essential-for-correct-cell-cycle-progress-in-trypanosoma-brucei
#15
Diane-Ethna Mbang-Benet, Yvon Sterkers, Lucien Crobu, Amélie Sarrazin, Patrick Bastien, Michel Pagès
BACKGROUND: Trypanosomatid parasites possess a single mitochondrion which is classically involved in the energetic metabolism of the cell, but also, in a much more original way, through its single and complex DNA (termed kinetoplast), in the correct progress of cell division. In order to identify proteins potentially involved in the cell cycle, we performed RNAi knockdowns of 101 genes encoding mitochondrial proteins using procyclic cells of Trypanosoma brucei. RESULTS: A major cell growth reduction was observed in 10 cases and a moderate reduction in 29 other cases...
2015: BMC Genomics
https://www.readbyqxmd.com/read/25066233/hierarchical-molecular-events-driven-by-oocyte-specific-factors-lead-to-rapid-and-extensive-reprogramming
#16
Jerome Jullien, Kei Miyamoto, Vincent Pasque, George E Allen, Charles R Bradshaw, Nigel J Garrett, Richard P Halley-Stott, Hiroshi Kimura, Keita Ohsumi, John B Gurdon
Nuclear transfer to oocytes is an efficient way to transcriptionally reprogram somatic nuclei, but its mechanisms remain unclear. Here, we identify a sequence of molecular events that leads to rapid transcriptional reprogramming of somatic nuclei after transplantation to Xenopus oocytes. RNA-seq analyses reveal that reprogramming by oocytes results in a selective switch in transcription toward an oocyte rather than pluripotent type, without requiring new protein synthesis. Time-course analyses at the single-nucleus level show that transcriptional reprogramming is induced in most transplanted nuclei in a highly hierarchical manner...
August 21, 2014: Molecular Cell
https://www.readbyqxmd.com/read/24810276/genome-sequencing-and-comparative-genomics-of-the-broad-host-range-pathogen-rhizoctonia-solani-ag8
#17
COMPARATIVE STUDY
James K Hane, Jonathan P Anderson, Angela H Williams, Jana Sperschneider, Karam B Singh
Rhizoctonia solani is a soil-borne basidiomycete fungus with a necrotrophic lifestyle which is classified into fourteen reproductively incompatible anastomosis groups (AGs). One of these, AG8, is a devastating pathogen causing bare patch of cereals, brassicas and legumes. R. solani is a multinucleate heterokaryon containing significant heterozygosity within a single cell. This complexity posed significant challenges for the assembly of its genome. We present a high quality genome assembly of R. solani AG8 and a manually curated set of 13,964 genes supported by RNA-seq...
May 2014: PLoS Genetics
https://www.readbyqxmd.com/read/24192459/serotonergic-neuron-regulation-informed-by-in-vivo-single-cell-transcriptomics
#18
Jennifer M Spaethling, David Piel, Hannah Dueck, Peter T Buckley, Jacqueline F Morris, Stephen A Fisher, Jaehee Lee, Jai-Yoon Sul, Junhyong Kim, Tamas Bartfai, Sheryl G Beck, James H Eberwine
Despite the recognized importance of the dorsal raphe (DR) serotonergic (5-HT) nuclei in the pathophysiology of depression and anxiety, the molecular components/putative drug targets expressed by these neurons are poorly characterized. Utilizing the promoter of an ETS domain transcription factor that is a stable marker of 5-HT neurons (Pet-1) to drive 5-HT neuronal expression of YFP, we identified 5-HT neurons in live acute slices. We isolated RNA from single 5-HT neurons in the ventromedial and lateral wings of the DR and performed single-cell RNA-Seq analysis identifying >500 G-protein coupled receptors (GPCRs) including receptors for classical transmitters, lipid signals, and peptides as well as dozens of orphan-GPCRs...
February 2014: FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology
https://www.readbyqxmd.com/read/23071390/a-next-generation-sequencing-approach-to-study-the-transcriptomic-changes-during-the-differentiation-of-physarum-at-the-single-cell-level
#19
Israel Barrantes, Jeremy Leipzig, Wolfgang Marwan
Physarum polycephalum is a unicellular eukaryote belonging to the amoebozoa group of organisms. The complex life cycle involves various cell types that differ in morphology, function, and biochemical composition. Sporulation, one step in the life cycle, is a stimulus-controlled differentiation response of macroscopic plasmodial cells that develop into fruiting bodies. Well-established Mendelian genetics and the occurrence of macroscopic cells with a naturally synchronous population of nuclei as source of homogeneous cell material for biochemical analyses make Physarum an attractive model organism for studying the regulatory control of cell differentiation...
2012: Gene Regulation and Systems Biology
https://www.readbyqxmd.com/read/22104983/non-laser-capture-microscopy-approach-for-the-microdissection-of-discrete-mouse-brain-regions-for-total-rna-isolation-and-downstream-next-generation-sequencing-and-gene-expression-profiling
#20
Norman Atkins, Charlie M Miller, Joseph R Owens, Fred W Turek
As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)(1) provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs(2-4). Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling(5) within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states...
2011: Journal of Visualized Experiments: JoVE
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