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SMARTer Citations

RNA-seq with intact cells and low-input samples

  1. Burns et al. (2015) Single-cell RNA-Seq resolves cellular complexity in sensory organs from the neonatal inner ear. Nat. Commun. 6:8557.

    The SMARTer Ultra Low Input RNA Kit for the Fluidigm C1 System was used to perform transcriptome analysis of 301 single cells from the utricular and cochlear sensory epithelia of newborn mice. Comparisons between auditory and vestibular cells from the respective tissues revealed divergent transcriptional profiles, despite the common origin shared by both cell types. The data provide important insights regarding developmental processes that give rise to distinct inner-ear cell types.
  2. Gray Camp et al. (2015) Human cerebral organoids recapitulate gene expression programs of fetal neocortex development. PNAS 112(51):15672–15677.

    The authors performed single-cell RNA-seq using the SMARTer Ultra Low Input RNA Kit for the Fluidigm C1 System to compare the transcriptional profiles of cells obtained from human cerebral organoids and fetal neocortex tissue. They found that cells in cortex-like regions of the organoids use very similar genetic programs to those found in fetal tissues to generate a structured cerebral cortex. The results suggest that cerebral organoids are a useful model system for studying aspects of human cortical development.
  3. Reichel, J., et al. (2015) Flow sorting and exome sequencing reveal the oncogenome of primary Hodgkin and Reed-Sternberg cells. Blood 125(7):1061–1072.

    Researchers optimized low-input exome sequencing of samples from patients with classical Hodgkin lymphoma (cHL) in order to investigate genetic alterations, including transcriptional regulation, which may characterize a tumor's microenvironment. Variants from exome data were validated by whole-transcriptome sequencing using the SMARTer Ultra Low Input RNA Kit for Sequencing with 1–5 ng total RNA extracted from flow-sorted Hodgkin and Reed-Sternberg (HRS) cells.

  4. Bao, J., et al. (2014) RAN-Binding Protein 9 is Involved in Alternative Splicing and is Critical for Male Germ Cell Development and Male Fertility. PLoS Genet. 10(12):e1004825.

    RNA immunoprecipitation followed by next-generation deep sequencing (RIP-Seq) and proteomic analyses was used to investigate the cellular functions of RANBP9 in the testicular somatic and spermatogenic cells of 6-week-old mice. The SMARTer Universal Low Input RNA Kit for Sequencing was used to synthesize 1–2 µg cDNA from pulled-down mRNAs, and subsequent Illumina library preparation was performed.

  5. Cabezas-Wallscheid, N., et al. (2014) Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis. Cell Stem Cell 15(4):507–522.

    This study used technological advances for analysis of rare cell populations to establish proteome, transcriptome, and genome-wide methylome data for hematopoietic stem cells (HSCs) and four multipotent progenitor (MPP) populations in order to better characterize changes in gene expression that occur at various stages of cell differentiation. cDNA libraries were generated from 10 ng total RNA using the SMARTer Ultra Low RNA Kit for Illumina Sequencing.

  6. Chen, L., et al. (2014) Transcriptional diversity during lineage commitment of human blood progenitors. Science 345(6204):1251033.

    This study looked at RNA-seq data from eight primary human hematopoietic progenitor populations representing various stages in the development of differentiated blood cells. The SMARTer Ultra Low RNA Kit for Illumina Sequencing was used to generate RNA-seq libraries from 25 poly(A)+ RNA samples, yielding 2.4 x 109 unique reads in total, ranging from 36–150 x 106 reads per sample.

  7. Heaton, N. H., et al. (2014) Long-term survival of influenza virus infected club cells drives immunopathology. J. Exp. Med. 211(9):17071714.

    Using the SMARTer Ultra Low RNA Kit for Illumina Sequencing to perform RNA-seq using club cells that survive infection with influenza A virus (IAV), the authors describe how these cells are transcriptionally unique from uninfected cells derived from a similar environment. The authors report that these club cells are important in preventing lung tissue damage caused by infection with IAV.

  8. Jenkins, N.T., et al. (2014) Transcriptome-wide RNA sequencing analysis of rat skeletal muscle feed arteries. I. Impact of obesity. J. Appl. Physiol. 116(8):1017–1032.

    This paper used next-generation RNA-seq technology to study the impact of obesity on global gene expression in skeletal muscle feed arteries. The SMARTer Ultra Low RNA Kit for Illumina Sequencing was used to generate full-length cDNA transcripts prior to Illumina library preparation. The authors found a total of 396 transcripts differentially expressed between aortic endothelial cell-enriched samples from obese and lean rats.

  9. Kim, S.T., et al. (2014) Transcriptome analysis of CD133-positive stem cells and prognostic value of surviving in colorectal cancer. Cancer Genomics Proteomics 11(5):256–266.

    The authors compared expression profiles of cell populations in primary and metastatic tumors from patients with colorectal cancer (CRC) in order to identify genes specific to CRC stem cells. Total RNA was isolated from CRC cells of each specimen, and RNA-seq libraries were generated with the SMARTer Ultra Low Input RNA for Illumina Sequencing kit.

  10. Patel, A.P., et al. (2014) Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science 344(6190):1396–1401.

    In this paper, researchers used single-cell RNA-seq, with the SMARTer Ultra Low RNA Kit for Illumina Sequencing, to probe transcriptional heterogeneity within primary glioblastomas. They found a high level of functional and transcriptional diversity between cells within the same tumor.

  11. Potier, D., et al. (2014) Mapping gene regulatory networks in Drosophila eye development by large-scale transcriptome perturbations and motif inference. Cell Rep. 9(6):2290–2303.

    In this study, RNA-seq was the first step in reverse engineering the gene regulatory networks of eye development in Drosophila. As listed in the supplementary material, the SMARTer Ultra Low RNA Kit for Illumina Sequencing was used for cDNA preparation from 0.5–3 µg total RNA extracted from various tissues of interest (EA disc, wing disc, brain) of Wandering 3rd instar larvae.

  12. Tang, X., et al. (2014) The eSNV-detect: a computational system to identify expressed single nucleotide variants from transcriptome sequencing data. Nucleic Acids Res. 42(22):e172.

    In this paper, the authors discussed the development of a novel computational system which calls and ranks variants from RNA-seq experiments, even those with low read depths. 16 single, live cells from the MDA-MB-231 breast cancer cell line were used in the analysis, with cDNAs prepared using the SMARTer Ultra Low RNA Kit for Illumina Sequencing. Where traditional multicellular RNA-seq data masked variants, single-cell mRNA-seq data showed heterogeneity among single cells, with 29 out of 31 candidate variants validated by Sanger sequencing.

  13. Ugale, A., et al. (2014) Hematopoietic stem cells are intrinsically protected against MLL-ENL-mediated transformation. Cell Reports 9(4):1246–1255.

    This paper describes the generation of a mouse model that allows conditional activation of the mixed-lineage leukemia/eleven-nineteen-leukemia (MLL-ENL) transcription factor in any cell type. This new model enables the study of differences in acute myeloid leukemia (AML) initiated from various hematopoietic progenitor subsets. Total RNA—from a total of 2,000 hematopoietic stem cells (HSCs) and precursors for granulocyte-macrophage progenitors (pGMs)—was used to generate RNA-seq libraries with the SMARTer Ultra Low RNA Kit for Illumina Sequencing. The authors found that inhibition of differentiation is a key early event during the narrow window for the development of leukemia.

  14. Brennecke, P., et al. (2013) Accounting for technical noise in single-cell RNA-seq experiments. Nature Methods 10(11):1093–1095.

    This study presents statistical methods to distinguish between biological variability and technical variability. Total RNA samples from mammalian and plant tissues were used as input for the SMARTer Ultra Low RNA Kit for Illumina Sequencing to validate the statistical approach.

  15. Henley, B.M., et al. (2013) Transcriptional regulation by nicotine in dopaminergic neurons. Biochem. Pharmacol. 86(8):1074–1083.

    This paper explores the possibility of a neuroprotective role of smoking in Parkinson’s disease. Twenty neurons were captured by LCM and cDNA was synthesized using the SMARTer Ultra Low RNA Kit for Illumina Sequencing. Each sequencing library generated >20 million uniquely mapped reads. This is the first known whole-transcriptome analysis of nicotine-treated substantia nigra pars compacta (SNc) neurons.

  16. Kadkhodaei, B., et al.(2013) Transcription factor Nurr1 maintains fiber integrity and nuclear-encoded mitochondrial gene expression in dopamine neurons. Proc Natl Acad Sci U S A. 110(6):2360–2365.

    In this study, researchers investigated the function of transcription factor Nurr1 in neuronal development and disease. Nurr1 is associated with features of Parkinson’s disease. mRNA-seq identified Nurr1-regulated genes and revealed its role in the activation of genes expressed in dopaminergic (DA) neurons. RNA extracted from laser-microdissected DA neurons of one-week-old mice was used with the SMARTer Ultra Low RNA Kit for Illumina Sequencing in order to generate cDNA libraries.

  17. Qiu, S., et al. (2013) Single-neuron RNA-seq: Technical feasibility and reproducibility. Front. Genet. 3:124.

    The authors report the development of protocols for single neuron RNA-seq coupled with electrophysiology. Following electrophysiological analysis, the contents of a single neuron from brain sections were extracted by aspiration with a thin glass electrode tip. cDNA libraries were generated using the SMARTer Ultra Low RNA Kit for Illumina Sequencing.

  18. Shalek, A.K., et al. (2013) Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells. Nature 498(7453):236–240.

    This study examines the response of individual bone marrow derived dendritic cells (BMDCs) following exposure to lipopolysaccharide (LPS). The authors used SMART-Seq technology, commercialized by Clontech in the SMARTer Ultra Low RNA Kit for Illumina Sequencing, to generate cDNA from single BMDCs.

  19. Yamaguchi, S., et al. (2013) Dynamics of 5-methylcytosine and 5-hydroxymethylcytosine during germ cell reprogramming. Cell Res. 23(3):329–339.

    This study investigated the dynamics of 5-methylcytosine (5mC) and its oxidative derivatives during primordial germ cell (PGC) reprogramming. The authors used the SMARTer Ultra Low RNA Kit for Illumina Sequencing for cDNA synthesis and amplification from total RNA purified from 300–5,000 sorted PGCs.

  20. Cann, G.M., et al. (2012) mRNA-seq of single prostate cancer circulating tumor cells reveals recapitulation of gene expression and pathways found in prostate cancer. PLoS ONE 7(11):e49144.

    This paper describes the use of MagSweeper technology to isolate circulating tumor cells (CTCs). The authors found that this isolation technique neither negatively impacts CTC survival nor alters the transcription profile of a prostate cancer cell line. The SMARTer Ultra Low RNA Kit for Illumina Sequencing was used to generate cDNA for single-cell mRNA-seq analysis of individual CTCs. While the quality of RNA extracted from individual cells varied, cDNA of sufficient quality and quantity for mRNA-seq was still able to be generated with this SMARTer Ultra Low kit.

  21. Hu, B.H., et al. (2012) Metalloproteinases and their associated genes contribute to the functional integrity and noise-induced damage in the cochlear sensory epithelium. J Neurosci. 32(43):14927–14941.

    The authors used gene expression analysis by RNA sequencing and qRT-PCR to investigate the role of matrix metalloproteinases (MMPs) and their related gene products in cochlear responses to traumatic noise injuries. RNA was extracted from cochlear sensory epithelia and pooled, and cDNA was synthesized with the SMARTer Ultra Low RNA Kit from 8–10 ng total RNA. The results suggest that MMPs may be useful therapeutic targets for noise-induced hearing loss.

  22. Ramsköld, D., et al. (2012) Full-length mRNA-seq from single cell levels of RNA and individual circulating tumor cells. Nature Biotechnol. 30(8):777–782.

    This paper describes the SMART-Seq™ method, which was the basis for the SMARTer Ultra Low kits for Illumina Sequencing. This method improved read coverage in comparison to previous methods for cDNA synthesis from single-cell level inputs, enabling the identification of SNPs and transcript isoforms.

    Note: Following the publication of this paper, a new cDNA synthesis kit for ultra-low input RNA and single cells has been developed. This kit facilitates the generation of higher quality RNA-seq data, especially for GC-rich genes.

High-throughput RNA-seq

  1. Shokhirev, M., et al. (2015) A multi-scale approach reveals that NF-KB cRel enforces a B-cell decision to divide. Mol Syst Biol. 11(2):783.

    High-throughput single-cell mRNA sequencing was used to complement flow cytometry and immunofluorescence microscopy in the development of a computational model simulating B-lymphocyte population dynamics in terms of the molecular networks within each cell. RNA-seq library generation was performed on individual B cells using the SMARTer Ultra Low RNA Kit for the Fluidigm C1 System.

  2. Videvall, E., et al. (2015) The avian transcriptome response to malaria infection. Mol Biol Evol. 32(5):1255–1267.

    The transcriptomic response of Eurasian siskins (Spinus spinus) infected with avian malaria parasites (Plasmodium ashfordi) was quantified via high-throughput RNA-seq. The SMARTer Ultra Low RNA Kit for Illumina Sequencing was used to generate libraries from total RNA extracted from whole-blood samples taken at various stages of infection.

  3. Brunskill, E.W., et al. (2014) Single cell dissection of early kidney development: multilineage priming. Development 141(15):3093–3101.

    The authors used single-cell RNA-seq to investigate seemingly uniform cell populations that give rise to distinct lineages in developing kidneys, starting with the definition of global expression profiles of 235 individual cells. The SMARTer Ultra Low RNA Kit for the Fluidigm C1 System was used to generate RNA-seq libraries from total mouse kidney single-cell suspensions, revealing single-cell specificity in RNA-processing patterns.

  4. Wu, A.R., et al. (2014) Quantitative assessment of single-cell RNA-sequencing methods. Nature Methods 11(1):41–46.

    In this study, researchers compared commercially available kits for cDNA synthesis from single cells. The authors present data in support of single-cell RNA-seq as a viable, reproducible method for quantitative transcriptome measurement. Both the SMARTer Ultra Low RNA Kit for Illumina Sequencing and the SMARTer Ultra Low RNA Kit for the Fluidigm C1 System were tested.

  5. Shalek, A.K., et al. (2013) Single-cell RNA-seq reveals dynamic paracrine control of cellular variation. Nature 510(7505):363–369.

    This paper looked at high-throughput single-cell transcriptomics to understand the extent, basis, and function of gene expression variation between cells that appear identical. The authors used the SMARTer Ultra Low RNA Kit for the Fluidigm C1 System for whole transcriptome amplification of over 1,700 single-cell RNA-seq libraries from primary mouse bone-marrow-derived dendritic cells under several different experimental conditions.

Comparison to other systems

  1. Shanker, S., et al. (2015) Evaluation of commercially available RNA amplification kits for RNA sequencing using very low input amounts of total RNA. J Biomol Tech. 26(1):4–18.

    This paper looked the accuracy and performance of commercial RNA-seq kits for picogram amounts of RNA. Four types of kits were used with three different concentrations (50 pg–5 ng) of commercially available RNA, and tested at multiple sites. Libraries produced with the SMARTer Ultra Low RNA Kit for Illumina Sequencing showed a substantially higher proportion of exonic reads than those produced with rRNA-depletion-based methods. This kit generated the highest percentage of unique reads and had the most consistent results across a dilution series.

  2. Alberti, A., et al. (2014) Comparison of library preparation methods reveals their impact on interpretation of metatranscriptomic data. BMC Genomics 15:912.

    This article compared sequencing metrics from libraries created from small amounts of a simplified mixture of bacterial total RNA. Four different cDNA synthesis and Illumina library preparation protocols were tested, including the SMARTer Stranded RNA-Seq Kit. The authors reported that for metatranscriptomic studies, the SMARTer Stranded RNA-Seq Kit performs very well in terms of library quality and yield.

  3. Adiconis, X., et al. (2013) Comparative analysis of RNA sequencing methods for degraded or low-input samples. Nature Methods 10(7):623–629.

    In this study, the authors systematically compared sequencing metrics generated with available cDNA synthesis methods using low-quality or low-quantity RNA as input. The authors reported that for low-quantity samples, the SMARTer Ultra Low RNA Kit for Illumina Sequencing generated cDNA for RNA-seq that resulted in a markedly lower percentage of reads map to rRNA and higher mapping to exons.

Other applications for RNA-seq

  1. Brown, B. A., et al. (2014) Seven strains of enterovirus D68 detected in the United States during the 2014 severe respiratory disease outbreak. Genome Announc. 2(6):e01201-14.

    Clinically relevant RNA from enterovirus D68 (EV-D68; a single-stranded, positive-sense RNA virus) was isolated either directly from nasopharyngeal swab supernatants or from inoculated rhabdomyosarcoma cells. This RNA was used as input for the SMARTer Universal Low Input RNA Library Prep Kit to reverse transcribe and then sequence EV-D68, resulting in one complete and six near-complete genomes.

     

Posters

  1. Bostick, M., et al. (2013) cDNA library generation for transcriptome analysis from total RNA equivalent to a single cell. J Biomol. Tech. 24(Suppl):S43.

    This poster highlights the excellent transcriptome data generated with the SMARTer Ultra Low method. The SMARTer Ultra Low RNA Kit for Illumina® Sequencing generated data that shows high correlation to MicroArray Quality Control (MAQC) data, even when generated from 0.1 ng of input RNA.

  2. Chitwood, J.L., et al. (2013) 131 RNA-seq transcriptome profiling of individual Rhesus macaque oocytes and pre-implantation embryos. Reprod. Fertil. Dev. 26(1):179–179.

    This poster describes the use of the SMARTer Ultra Low RNA Kit for Illumina Sequencing for transcriptome analysis of Rhesus macaque oocytes and embryos.

  3. Zueckert-Gaudenz, K., et al. (2013) Evaluation of whole transcriptome amplification methods by RNA-seq. J. Biomol. Tech. 24(Suppl):S57–S58.

    In this study, the SMARTer Ultra Low RNA Kit for Illumina Sequencing was compared to three other commercially available kits for low-input RNA-seq. cDNA libraries generated from total RNA with this Clontech kit performed well across all criteria tested.

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