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Smart seq ultra low input rna kit

Manufactured by Takara Bio
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Sourced in United Kingdom, Japan

The SMART-Seq Ultra Low Input RNA kit is a solution for RNA sequencing from low input samples. It enables cDNA synthesis and amplification from small amounts of total RNA.

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Lab products found in correlation

Nextseq 500, by Illumina (4 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions) Hiseq 2500 sequencer, by Illumina (2 mentions) Nextseq 500 550 mid output kit v2, by Illumina (2 mentions) Nextera xt dna library prep kit, by Illumina (2 mentions) Nucleospin rna xs kit, by Macherey-Nagel (2 mentions) Hiseq machine, by Illumina (2 mentions) Rnaqueous kit, by Thermo Fisher Scientific (2 mentions) Nextera xt dna library kit, by Illumina (2 mentions) 2100 bioanalyzer, by Agilent Technologies (2 mentions) Prism software, by GraphPad (1 mentions) Hiseq 3000 sequencer, by Illumina (1 mentions) Rna isolation kit, by Macherey-Nagel (1 mentions) Nextra xt kit, by Illumina (1 mentions) Hiseq3000 genome analyzer, by Illumina (1 mentions) Ovation rna seq system v2, by Tecan (1 mentions) Dnase 1, by Thermo Fisher Scientific (1 mentions) Rna clean and concentrator kit, by Zymo Research (1 mentions) Novaseq 6000, by Illumina (1 mentions) On column dnase digestion kit, by Qiagen (1 mentions)

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RNA‐Seq (2 citations)

16 protocols using smart seq ultra low input rna kit

1

RNA Quantification from Laser Captured Samples

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RNA from laser-captured samples was extracted using the RNeasy Micro Kit. Genomic DNA was removed by on-column DNase I digestion. RNA amount and quality was monitored by an Agilent bioanalyzer. Five hundred picograms of RNA was used for cDNA amplification using the SMART-Seq Ultra Low Input RNA Kit (Clontech). Quantitative PCR was performed using amplified cDNA on an Opticon Real-Time PCR machine. iQ qPCR (Bio-Rad) mastermix was used for all of the reactions. RNA expression levels were normalized to β2-microglobulin mRNA levels. Primer specificity and genomic RNA contamination was monitored using the melting curve of the PCR products. Primer sequences were obtained from the Origene validated quantitative PCR (qPCR) primer collection (Origene Technologies; Table 1). Statistical analysis was performed with Prism software (GraphPad Software). The two groups were compared using an unpaired nonparametric Mann–Whitney test, and significance was defined as p < 0.05 (*).
Chatzi C., Zhang Y., Shen R., Westbrook G.L, & Goodman R.H. (2016). Transcriptional Profiling of Newly Generated Dentate Granule Cells Using TU Tagging Reveals Pattern Shifts in Gene Expression during Circuit Integration,. eNeuro, 3(1), ENEURO.0024-16.2016.
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2

Single-cell RNA-seq of B cells

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Total RNA was obtained by sorting antigen-specific B cells (350–1,000 total cells) ex vivo directly into the 100 μl of cell lysis buffer provided in the RNA isolation kit (Macherey-Nagel, RNA-XS). Further, cDNA libraries were synthesized using the commercially available SMART-Seq Ultra Low Input RNA kit (Clontech), as per the manufacturer’s protocols. After preparation of cDNA libraries, they were first tagmented and then barcoded by indexing primers using the Nextra XT kit (Illumina). The libraries were pooled and a 76bp paired-end sequencing was performed on an Illumina HiSeq3000 sequencer to yield a minimum of 17.4 million reads per library (range = 17.4 – 37.3 million).
Mahendra A., Yang X., Abnouf S., Adolacion J.R., Park D., Soomro S., Roszik J., Coarfa C., Romain G., Wanzeck K., Bridges SL J.r., Aggarwal A., Qiu P., Agarwal S.K., Mohan C, & Varadarajan N. (2019). Beyond autoantibodies: Biological roles of human autoreactive B cells in rheumatoid arthritis revealed by RNA-sequencing.. Arthritis & rheumatology (Hoboken, N.J.), 71(4), 529-541.
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3

RNA Isolation and RNA-seq Library Preparation

Cited 1 time
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Three replicate samples for each genotype were collected, and total RNA was isolated using Trizol LS then treated with DNase I (Life Technologies). RNA was column-purified using the RNA Clean and Concentrator Kit (Zymo Research) and previously published protocols (Choi et al. 2012 (link)). RNA integrity was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA), and samples with an RNA integrity number (RIN) of 7.0 or greater were used for RNA-Seq. All samples were amplified using the SMART-Seq Ultra Low Input RNA Kit (TAKARA/Clontech) at 10 cycles, except for the Neurog3GFP/+ and Neurog3GFP/GFP samples, which were prepared using the Ovation RNA-Seq System V2 (NuGEN). cDNA was prepared using the Low Input Library Kit (Clontech) and sequenced using either an Illumina HiSeq3000 genome analyzer to obtain paired-end, 75-bp reads, or Illumina NovaSeq6000 to get paired-end, 100-bp reads. RNA samples were sequenced to an average depth of ∼5.0 × 107 reads. RNA-Seq data have been deposited in ArrayExpress (accession no. E-MTAB-10262).
Dudek K.D., Osipovich A.B., Cartailler J.P., Gu G, & Magnuson M.A. (2021). Insm1, Neurod1, and Pax6 promote murine pancreatic endocrine cell development through overlapping yet distinct RNA transcription and splicing programs. G3: Genes|Genomes|Genetics, 11(11), jkab303.
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4

Transcriptome Profiling of Human Adipose Tissue

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Total RNA was extracted and purified from human fat specimens using a Qiagen RNeasy Micro kit (Qiagen), and residual genomic DNA was further removed by an on-column DNase digestion kit (Qiagen). Library construction, sequencing, and data analysis were performed at the Center for Cancer Computational Biology Core Facilities at Dana-Farber Cancer Institute (DFCI). Sequencing libraries were prepared using a SMART-Seq Ultra Low Input RNA kit (Clontech). The resulting library size distributions were analyzed using a Bioanalyzer (Agilent). The concentration of the library was determined using a DNA High-Sensitivity Qubit assay, and the final functional library concentration was determined through qPCR using Illumina adaptor-specific primers with a KAPA SYBR FAST Universal qPCR kit (Sigma–Aldrich).The library pools were loaded at final concentrations of 2 pM on single-read 75 flow cells and sequenced on an Illumina NextSeq 500 platform. Sequencing reads were aligned to the reference genome (Ensembl GRCh37.75) using the RNA-specific STAR aligner (v2.3.1z4).
Fazeli P.K., Zhang Y., O'Keefe J., Pesaresi T., Lun M., Lawney B, & Steinhauser M.L. (2020). Prolonged fasting drives a program of metabolic inflammation in human adipose tissue. Molecular Metabolism, 42, 101082.
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5

Sequencing of Single-Cell cDNA Libraries

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The cDNA libraries for used for sequencing of 4 total RNA samples were synthesized using a SMART-Seq Ultra Low Input RNA kit (Takara) in the OHSU Massively Parallel Sequencing Shared Resource Core Facility. Two of the cDNA libraries contained tdTomato+/GFP+ cells (S3-Gbx2+ ACs) and two samples contained the cDNA libraries for tdTomato+ only cells (S5-Gbx2+ ACs). Quality and quantity of the cDNA libraries was determined on a Bioanalyzer. For multiplex sequencing, all four cDNA sample libraries were loaded on a single lane on a HiSeq 2500 sequencer (Illumina). Libraries were sequenced to a depth of 45–55 million reads per sample. Alignment rate of total reads was >97% across all samples.
Kerstein P.C., Leffler J., Sivyer B., Taylor W.R, & Wright K.M. (2020). Gbx2 Identifies Two Amacrine Cell Subtypes with Distinct Molecular, Morphological, and Physiological Properties. Cell reports, 33(7), 108382.
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6

Single-cell RNA-seq of Migratory Cells

Cited 2 times
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RNA was isolated from samples of 1000 migratory or unsorted cells in triplicate using the Nucleospin RNA XS kit (Macherey-Nagel). cDNA libraries were amplified using the SMART-seq ultra low input RNA kit (Takara) and then tagmented and barcoded by indexing primers using Nextera XT DNA library prep kit (Illumina). Samples were pooled and paired-end sequenced on an Illumina NextSeq 500 using the NextSeq 500/550 Mid Output v2 kit with 150 cycles and an output of up to 130 million reads. After quality control of raw data using Illumina pipeline, RNA-seq reads were mapped to hg38 reference genome using HISAT253 (link) aligner. Htseq-count command from the HTSeq framework54 (link) was used to quantify read counts per gene from aligned reads using human ENSEMBL 86 (GRCh38.p7) gene models. The Bioconductor/R package DESeq255 (link) was used for normalization and differential gene expression analysis. Pathway analysis and gene ontology clustering were performed using the Database for Annotation, Visualization, and Integrated Discover (DAVID)56 (link),57 (link).
Yankaskas C.L., Thompson K.N., Paul C.D., Vitolo M.I., Mistriotis P., Mahendra A., Bajpai V.K., Shea D.J., Manto K.M., Chai A.C., Varadarajan N., Kontrogianni-Konstantopoulos A., Martin S.S, & Konstantopoulos K. (2019). A microfluidic assay for the quantification of the metastatic propensity of breast-cancer specimens. Nature biomedical engineering, 3(6), 452-465.
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7

RNA-Seq of Chicken and Lamprey Embryos

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Chicken libraries were prepared using the SMART-Seq Ultra Low Input RNA Kit (Takara) according to the manufacturer’s protocol. For lamprey embryos, tissue was dissected from the cranial dorsal neural tubes of n=100 T21 and trunk neural tube of n=100 T23.5 embryos. Total RNA was extracted using the RNAqueous kit (Ambion). RNA-Seq was performed at the Millard and Muriel Jacobs Genetics and Genomics Laboratory (California Institute of Technology, Pasadena, CA) at 50 million reads on 2 biological replicates for both the T21 cranial and T23.5 trunk neural tube samples. Sequencing libraries were built according to Illumina Standard Protocols. SR50 sequencing was performed in a HiSeq Illumina machine. Databases have been deposited to NCBI (BioProject # PRJNA497902)
Martik M.L., Gandhi S., Uy B.R., Gillis J.A., Green S.A., Simoes-Costa M, & Bronner M.E. (2019). Evolution of the New Head by gradual acquisition of neural crest regulatory circuits. Nature, 574(7780), 675-678.
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8

SMART-Seq® Ultra® Low Input RNA Sequencing

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A SMART-Seq® Ultra® Low Input RNA Kit was used to synthesize the full cDNA from 1000 MCs according to the manufacturer’s instructions (Takara Bio, London, UK). The analysis was performed on the next-generation sequencing Illumina HiSeq platform (Illumina, Cambridge, UK).
West P.W., Tontini C., Atmoko H., Kiss O., Garner T., Bahri R., Warren R.B., Griffiths C.E., Stevens A, & Bulfone-Paus S. (2023). Human Mast Cells Upregulate Cathepsin B, a Novel Marker of Itch in Psoriasis. Cells, 12(17), 2177.
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9

RNA-Seq of Chicken and Lamprey Embryos

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Chicken libraries were prepared using the SMART-Seq Ultra Low Input RNA Kit (Takara) according to the manufacturer’s protocol. For lamprey embryos, tissue was dissected from the cranial dorsal neural tubes of n=100 T21 and trunk neural tube of n=100 T23.5 embryos. Total RNA was extracted using the RNAqueous kit (Ambion). RNA-Seq was performed at the Millard and Muriel Jacobs Genetics and Genomics Laboratory (California Institute of Technology, Pasadena, CA) at 50 million reads on 2 biological replicates for both the T21 cranial and T23.5 trunk neural tube samples. Sequencing libraries were built according to Illumina Standard Protocols. SR50 sequencing was performed in a HiSeq Illumina machine. Databases have been deposited to NCBI (BioProject # PRJNA497902)
Martik M.L., Gandhi S., Uy B.R., Gillis J.A., Green S.A., Simoes-Costa M, & Bronner M.E. (2019). Evolution of the New Head by gradual acquisition of neural crest regulatory circuits. Nature, 574(7780), 675-678.
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10

Sequencing of Single-Cell cDNA Libraries

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The cDNA libraries for used for sequencing of 4 total RNA samples were synthesized using a SMART-Seq Ultra Low Input RNA kit (Takara) in the OHSU Massively Parallel Sequencing Shared Resource Core Facility. Two of the cDNA libraries contained tdTomato+/GFP+ cells (S3-Gbx2+ ACs) and two samples contained the cDNA libraries for tdTomato+ only cells (S5-Gbx2+ ACs). Quality and quantity of the cDNA libraries was determined on a Bioanalyzer. For multiplex sequencing, all four cDNA sample libraries were loaded on a single lane on a HiSeq 2500 sequencer (Illumina). Libraries were sequenced to a depth of 45–55 million reads per sample. Alignment rate of total reads was >97% across all samples.
Kerstein P.C., Leffler J., Sivyer B., Taylor W.R, & Wright K.M. (2020). Gbx2 Identifies Two Amacrine Cell Subtypes with Distinct Molecular, Morphological, and Physiological Properties. Cell reports, 33(7), 108382.
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