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SMARTer RNA-seq citations
Whole transcriptome profiling from single-cell or ultra-low-input samples (dT primed)
Cheng, L. et al. Blocking type I interferon signaling enhances T cell recovery and reduces HIV-1 reservoirs. J. Clin. Invest. 127, 269–279 (2017).
This study suggests that blocking the IFN-α/β receptor (IFNAR) may provide a potential strategy to enhance immune recovery and reduce HIV-1 reservoirs in individuals with sustained elevations in IFN-I signaling during suppressive combined antiretroviral therapy (cART). cDNA was prepared using the SMART-Seq v4 Ultra Low RNA-Seq Kit for Sequencing from RNA purified from human CD8 T cells from spleens of humanized mice.
Paul, A. L. et al. Genetic dissection of the Arabidopsis spaceflight transcriptome: Are some responses dispensable for the physiological adaptation of plants to spaceflight? PLoS One 12, e0180186 (2017).
This study examined the influence of genotype on the physiological adaptation to spaceflight as measured by gene expression patterns. RNA-seq transcriptome profiling of cDNA libraries constructed with the SMART-Seq V4 Ultra Low Input RNA Kit for Sequencing provided the metric by which to measure the impact of the environment and to assess whether it is possible to eliminate all or part of the spaceflight response by manipulating the genotype of the plant. The primary conclusion of this work is that much of the plant response to spaceflight is potentially nonadaptive in that at least most of the gene expression responses could be genetically eliminated without overtly compromising growth in space.
Sugiyama, H. et al. Nat1 promotes translation of specific proteins that induce differentiation of mouse embryonic stem cells. Proc. Natl. Acad. Sci. U. S. A. 114, 340–345 (2017).
The authors have demonstrated that Nat1, which is homologous to the C-terminal two-thirds of eukaryotic translation initiation factor 4G, is involved in the translation of proteins that are required for the differentiation of mouse embryonic stem cells. The qRT-PCR single-cell expression analysis performed in this study used cDNAs preamplified using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing.
Burns, G. W., Brooks, K. E. & Spencer, T. E. Extracellular Vesicles Originate from the Conceptus and Uterus During Early Pregnancy in Sheep. Biol. Reprod. 94, 56 (2016).
The identification of extracellular vesicles (EVs) as a component of ovine uterine luminal fluid raises the possibility that they are novel mediators of conceptus-maternal interactions. Using SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing and the Low Input Library Prep Kit, libraries were constructed from the EVs of Day 14 cultured conceptuses. These studies support the ideas that EVs emanate from both the conceptus trophectoderm and uterine epithelia and are involved in intercellular communication between those cells during the establishment of pregnancy in sheep.
DeLaughter, D. M. et al. Single-Cell Resolution of Temporal Gene Expression during Heart Development. Dev. Cell 39, 480–490 (2016).
To characterize lineage-specific, spatiotemporal developmental programs, the authors performed single-cell RNA sequencing of >1,200 murine cells isolated at seven timepoints spanning embryonic day 9.5 (primordial heart tube) to postnatal day 21 (mature heart). The data, generated with cDNAs libraries prepared using the SMARTer Ultra Low RNA Kit for Illumina Sequencing, allowed the definition of distinct developmental ages of human and mouse pluripotent stem cell-derived cardiomyocytes and characterized lineage-specific maturation defects in hearts of mice with heterozygous mutations.
Lau, C. M. et al. Leukemia-associated activating mutation of Flt3 expands dendritic cells and alters T cell responses. J. Exp. Med. 213, 415–31 (2016).
This paper studied the internal tandem duplication (ITD) in FLT3, the receptor for cytokine FLT3 ligand (FLT3L), which is a common genetic alteration in acute myeloid leukemia. Constitutively active FLT3-ITD promotes the expansion of transformed progenitors but also has pleiotropic effects on hematopoiesis. cDNA libraries from total RNA isolated from splenocytes (CD8+ cDCs, CD11b+ cDCs, pDCs) were prepared using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing. The authors determined that this mutation directly affects dendritic cell development, indirectly modulating T-cell homeostasis and supporting Treg-cell expansion, and they hypothesized that this effect of FLT3-ITD might impair immunosurveillance and promote leukemogenesis in a cell-extrinsic manner.
Loh, K. M. et al. Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types. Cell 166, 451–467 (2016).
This study mapped the bifurcating lineage choices leading from pluripotency to twelve human mesodermal lineages, including bone, muscle, and heart. Single-cell libraries were prepared with the SMARTer Ultra Low RNA Kit for Illumina Sequencing. The authors defined the extrinsic signals controlling each binary lineage decision, enabling them to logically block differentiation towards unwanted fates and rapidly steer pluripotent stem cells towards 80–99% pure human mesodermal lineages at most branch points. This strategy enabled the generation of human bone and heart progenitors that could engraft in respective in vivo models.
Burns, J. C., Kelly, M. C., Hoa, M., Morell, R. J. & Kelley, M. W. Single-cell RNA-Seq resolves cellular complexity in sensory organs from the neonatal inner ear. Nat. Commun. 6, 8557 (2015).
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.
Camp, J. G. et al. Human cerebral organoids recapitulate gene expression programs of fetal neocortex development. Proc. Natl. Acad. Sci. U. S. A. 112, 15672–156777 (2015).
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.
Reichel, J. et al. Flow sorting and exome sequencing reveal the oncogenome of primary Hodgkin and Reed-Sternberg cells. Blood 125, 1061–1072 (2015).
Researchers optimized low-input exome sequencing of samples from patients with classical Hodgkin lymphoma (cHL) to investigate genetic alterations, including transcriptional regulation, which may characterize the tumor 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.
Shokhirev, M. N. et al. A multi-scale approach reveals that NF-κB cRel enforces a B-cell decision to divide. Mol. Syst. Biol. 11, 783 (2015).
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.
Videvall, E., Cornwallis, C. K., Palinauskas, V., Valkiunas, G. & Hellgren, O. The avian transcriptome response to malaria infection. Mol. Biol. Evol. 32, 1255–1267 (2015).
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.
Brunskill, E. W. et al. Single cell dissection of early kidney development: multilineage priming. Development 141, 3093–3101 (2014).
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.
Cabezas-Wallscheid, N. et al. Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis. Cell Stem Cell 15, 507–522 (2014).
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 of total RNA using the SMARTer Ultra Low RNA Kit for Illumina Sequencing.
Chen, L. et al. Transcriptional diversity during lineage commitment of human blood progenitors. Science 345, 1251033 (2014).
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 polyA+ RNA samples, yielding 2.4 x 109 unique reads in total, ranging from 36–150 x 106 reads per sample.
Heaton, N. S. et al. Long-term survival of influenza virus infected club cells drives immunopathology. J. Exp. Med. 211, 1707–1714 (2014).
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.
Jenkins, N. T. et al. Transcriptome-wide RNA sequencing analysis of rat skeletal muscle feed arteries. I. Impact of obesity. J. Appl. Physiol. 116, 1017–1032 (2014).
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.
Kim, S. T. et al. Transcriptome analysis of CD133-positive stem cells and prognostic value of survivin in colorectal cancer. Cancer Genomics and Proteomics 11, 259–266 (2014).
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 Kit RNA for Illumina Sequencing.
Patel, A. P. et al. Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science 344, 1396–1401 (2014).
In this study, 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.
Potier, D. et al. Mapping Gene Regulatory Networks in Drosophila Eye Development by Large-Scale Transcriptome Perturbations and Motif Inference. Cell Rep. 9, 2290–2303 (2014).
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 of total RNA extracted from various tissues of interest (EA disc, wing disc, brain) of Wandering 3rd instar larvae.
Shalek, A. K. et al. Single-cell RNA-seq reveals dynamic paracrine control of cellular variation. Nature 510, 363–369 (2014).
This study 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.
Tang, X. et al. The eSNV-detect: A computational system to identify expressed single nucleotide variants from transcriptome sequencing data. Nucleic Acids Res. 42, e172 (2014).
In this study, 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.
Ugale, A. et al. Hematopoietic Stem Cells Are Intrinsically Protected against MLL-ENL-Mediated Transformation. Cell Rep. 9, 1246–1255 (2014).
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 and precursors for granulocyte-macrophage progenitors—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.
Wu, A. R. et al. Quantitative assessment of single-cell RNA-sequencing methods. Nat. Methods 11, 41–46 (2014).
In this study, the authors 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.
Brennecke, P. et al. Accounting for technical noise in single-cell RNA-seq experiments. Nat. Methods 10, 1093–1095 (2013).
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.
Henley, B. M. et al. Transcriptional regulation by nicotine in dopaminergic neurons. Biochem. Pharmacol. 86, 1074–1083 (2013).
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.
Kadkhodaei, B. et al. Transcription factor Nurr1 maintains fiber integrity and nuclear-encoded mitochondrial gene expression in dopamine neurons. Proc. Natl. Acad. Sci. U. S. A. 110, 2360–2365 (2013).
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.
Shalek, A. K. et al. Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells. Nature 498, 236–240 (2013).
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 Takara Bio in the SMARTer Ultra Low RNA Kit for Illumina Sequencing, to generate cDNA from single BMDCs.
Yamaguchi, S. et al. Dynamics of 5-methylcytosine and 5-hydroxymethylcytosine during germ cell reprogramming. Cell Res. 23, 329–339 (2013).
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.
Cann, G. M. et al. mRNA-Seq of Single Prostate Cancer Circulating Tumor Cells Reveals Recapitulation of Gene Expression and Pathways Found in Prostate Cancer. PLoS One 7, e49144 (2012).
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 transcriptional 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 could still be generated with this SMARTer Ultra Low kit.
Hu, B. H. et al. Metalloproteinases and their associated genes contribute to the functional integrity and noise-induced damage in the cochlear sensory epithelium. J. Neurosci. 32, 14927–14941 (2012).
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 for Illumina Sequencing from 8–10 ng of total RNA. The results suggest that MMPs may be useful therapeutic targets for noise-induced hearing loss.
Ramsköld, D. et al. Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nat. Biotechnol. 30, 777–782 (2012).
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. Following the publication of this paper, a new cDNA synthesis kit for ultra-low input RNA and single cells was developed. This kit facilitates the generation of higher-quality RNA-seq data, especially for GC-rich genes.
Qiu, S. et al. Single-neuron RNA-Seq: Technical feasibility and reproducibility. Front. Genet. 3, 124 (2012).
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.
Whole transcriptome profiling from single-cell or ultra-low-input samples (randomly primed)
Huang, A. C. et al. T-cell invigoration to tumour burden ratio associated with anti-PD-1 response. Nature 545, 60–65 (2017).
CD8 T cells can mount responses against many human cancer types, especially those with higher mutational burden. The authors used immune profiling of peripheral blood from patients with stage IV melanoma before and after treatment with the programmed cell death 1 (PD1)-targeting antibody, identifying a clinically accessible potential on-treatment predictor of response to PD-1 blockade. RNA-seq libraries were prepared from CD8 T cells using the SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian and examined for changes in transcript expression following treatment.
Sagara, A. et al. Endocan as a prognostic biomarker of triple-negative breast cancer. Breast Cancer Res. Treat. 161, 269–278 (2017).
This study indicates that endocan could be used as a blood-based prognostic biomarker in triple-negative breast cancer patients. Total RNA was isolated from each cell line, and libraries for RNA-seq were constructed using the SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian.
Sagara, A. et al. Intrinsic Resistance to 5-Fluorouracil in a Brain Metastatic Variant of Human Breast Cancer Cell Line, MDA-MB-231BR. PLoS One 11, e0164250 (2016).
The authors showed that MDA-MB-231BR, a metastatic brain variant of a human breast cancer cell line, was refractory to treatment with 5-fluorouracil (5-FU)-even without chronic drug exposure-as compared to its parent cell line (MDA-MB-231) and a bone metastatic variant (MDA-MB-231SCP2). These results indicate that BCL2A1 is a key contributor to the intrinsic 5-FU-resistance in MDA-MB-231BR. Libraries for RNA-seq were constructed using SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian.
Bao, J. et al. RAN-Binding Protein 9 is Involved in Alternative Splicing and is Critical for Male Germ Cell Development and Male Fertility. PLoS Genet. 10, e1004825 (2014).
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.
Dadonaite, B., Barilaite, E., Fodor, E., Laederach, A. & Bauer, D. L. The structure of the influenza A virus genome. bioRxiv 236620 (2017). doi:10.1101/236620
The researchers utilized the SMARTer smRNA-Seq Kit for Illumina to investigate RNA-RNA interactions inside influenza virions. This study provides details on how influenza virus controls its packaging and growth, as well as the mechanism of potential reassortment of genomes among different strains. The article discusses how this data can be used to predict the emergence of new pandemic influenza strains.
Hornstein, N. et al. Ligation-free ribosome profiling of cell type-specific translation in the brain. Genome Biol. 17, 149 (2016).
In this study, the authors developed a method for ligation-free ribosomal profiling using the SMARTer smRNA-Seq Kit for Illumina. They used this approach with mouse brain tissue to identify targets of mTOR signaling in the brain.
Griswold, A. J. et al. Transcriptomic analysis of synovial extracellular RNA following knee trauma: A pilot study. J. Orthop. Res. (2017). doi:10.1002/jor.23802
Extracellular RNA (exRNA) from synovial fluids of post-traumatic knee injury patients was profiled using the SMARTer smRNA-Seq Kit for Illumina. The study identified several matrix metalloproteinases by analyzing exRNA fragments in synovial fluid samples obtained from patients.
Pasquariello, R. et al. Micro-RNA sequencing of individual human oocytes. Fertil. Steril. 108, e144 (2017).
In this study, the researchers performed miRNA profiling on human MII oocytes using the SMARTer smRNA-Seq Kit for Illumina. The article discusses a novel methodology for profiling individual oocytes and suggests an application using miRNAs as markers for reproductive failure.
Alberti, A. et al. Comparison of library preparation methods reveals their impact on interpretation of metatranscriptomic data. BMC Genomics (2014). doi:10.1186/1471-2164-15-912
The authors compared four library preparation methods for the interpretation of metatranscriptomic data: SMARTer Stranded RNA-Seq Kit (Takara Bio), Ovation Encore Complete Prokaryotic (NuGEN), Ovation RNA-Seq v2 (NuGEN), and TruSeq® Stranded Total RNA (Illumina). They found the SMARTer Stranded RNA-Seq Kit to be the best solution for smaller amounts of RNA, as it was the fastest, least biased, and most reproducible method for low-input mixed microbial samples.
de Oliveira, L. et al. Transcriptomic analysis of the red seaweed Laurencia dendroidea (Florideophyceae, Rhodophyta) and its microbiome. BMC Genomics 13, 487 (2012).
The authors performed simultaneous RNA-seq on seaweed and its microbiome and showed the importance of the red seaweed's microbiome in the breakdown of organic matter and production of nitrogen. The seaweed transcriptome also contained transcripts related to terpene biosynthesis, a building block for a variety of functionally active small molecules. The use of the SMARTer Stranded RNA-Seq Kit enabled interrogation of both the algae and the microbiome in the same preparation.
Hughes, L. C. et al. Transcriptomic differentiation underlying marine-to-freshwater transitions in the South American silversides Odontesthes argentinensis and O. bonariensis (Atheriniformes). Ecol. Evol. 7, 5258–5268 (2017).
The authors studied the transcriptome and microbiome differences in fish species that move from the ocean to inland rivers (marine to freshwater transitions). Few species are able to tolerate the extreme change in salt concentration between ocean water and fresh water. This study characterized the differences in transcriptional profiles and microbiome content of two species that make this remarkable transition. The team of researchers were able to simultaneously interrogate both the fish transcriptome and the microbiome of the fish gill in the same prep and sequencing experiment. The random priming approach of the SMARTer Stranded RNA-Seq Kit enabled this study.
Marynowska, M. et al. Optimization of a metatranscriptomic approach to study the lignocellulolytic potential of the higher termite gut microbiome. BMC Genomics 18, 681 (2017).
The authors used two different microbe transcript enrichment approaches to examine the microbiome content of the termite gut. They optimized a workflow to isolate RNA and prepare libraries from a very challenging sample type: the termite gut microbiome, which plays a critical role in breaking down complex molecules from plant matter. The SMARTer Stranded RNA-Seq Kit enabled use of low inputs of enriched RNA, with excellent transcriptome representation.
Sureshchandra, S. et al. Inflammatory determinants of pregravid obesity in placenta and peripheral blood. Front. Physiol. 9, 1089 (2018).
The researchers compared maternal cytokines and placental gene expression in lean and obese pregnant women in order to understand the biological underpinnings of a higher rate of complications in obese pregnant women. They looked at the levels of immune system signaling molecules (cytokines and growth factors), maternal gene expression, and placental microbiome gene expression. The study helped better connect the impact of obesity on poor pregnancy outcomes by linking expressed genes with the small molecules that act to signal immune responses. The study helped support the hypothesis that pregravid obesity is associated with a heightened systemic inflammation and dysregulation of nutrient transport for the fetus. For gene expression, they were able to simultaneously interrogate both the maternal gene expression profile and the microbiome transcriptome in a single library prep with the SMARTer Stranded RNA-Seq system. Maternal ribosomal RNA was depleted using a RiboGone kit.
Comparison to other systems
Shanker, S. et al. Evaluation of commercially available RNA amplification kits for RNA sequencing using very low input amounts of total RNA. J. Biomol. Tech. 26, 4–18 (2015).
This paper looked at 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.
Alberti, A. et al. Comparison of library preparation methods reveals their impact on interpretation of metatranscriptomic data. BMC Genomics 15, 912 (2014).
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.
Adiconis, X. et al. Comparative analysis of RNA sequencing methods for degraded or low-input samples. Nat. Methods 10, 623–629 (2013).
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
Brown, B. A., et al. Seven Strains of Enterovirus D68 Detected in the United States during the 2014 Severe Respiratory Disease Outbreak. Genome Announc. 2, 6336 (2014).
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.
Bostick, M. et al. cDNA library generation for transcriptome analysis from total RNA equivalent to a single cell. J. Biomol. Tech. 24(Suppl), S43 (2013).
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.
Chitwood, J.L. et al. RNA-seq transcriptome profiling of individual Rhesus macaque oocytes and pre-implantation embryos. Reprod. Fertil. Dev. 26, 179 (2013).
This poster describes the use of the SMARTer Ultra Low RNA Kit for Illumina Sequencing for transcriptome analysis of Rhesus macaque oocytes and embryos.
Zueckert-Gaudenz, K. et al. Evaluation of whole transcriptome amplification methods by RNA-seq. J. Biomol. Tech. 24(Suppl), S57–S58 (2013).
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 the SMARTer kit performed well across all criteria tested.
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