Low input library prep kit

Manufactured by Takara Bio

Sourced in United States

The Low Input Library Prep Kit is a laboratory tool designed for the preparation of DNA libraries from small input amounts. It provides a streamlined workflow for generating libraries suitable for next-generation sequencing applications.

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Lab products found in correlation

2100 bioanalyzer, by Agilent Technologies (6 mentions) Smart seq v4 ultra low input rna kit for sequencing, by Takara Bio (4 mentions) Rneasy micro kit, by Qiagen (4 mentions) Nextseq 550 system, by Illumina (2 mentions) Smarter ultra low input rna kit for sequencing v3, by Takara Bio (2 mentions) High sensitivity dna kit, by Agilent Technologies (2 mentions) Picopure kit, by Thermo Fisher Scientific (2 mentions) Hiseq 2500 sequencer, by Illumina (2 mentions) Smarter ultra low input rna for sequencing hv kit, by Illumina (2 mentions) Novaseq 6000, by Illumina (1 mentions) Kapa s library quantification kit, by Roche (1 mentions) 2200 tapestation system, by Agilent Technologies (1 mentions) Ribo zero magnetic gold kit, by Illumina (1 mentions) High sensitivity d1000 screentape, by Agilent Technologies (1 mentions) High sensitivity d1000 reagents, by Agilent Technologies (1 mentions) Rna 6000 pico labchip, by Agilent Technologies (1 mentions) Smarter universal low input rna kit for sequencing, by Takara Bio (1 mentions) Nucleospin totalrna ffpe xs kit, by Macherey-Nagel (1 mentions) Hiseq 2000 system, by Illumina (1 mentions) S2 system, by Covaris (1 mentions)

Close spelling variants of this product

Smarter Low Input and Low input library prep kits (2 citations)

17 protocols using low input library prep kit

RNA-Seq Analysis of Mouse Samples

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RNA samples were analyzed using an Agilent 2100 Bioanalyzer, and only those samples with an RNA integrity number of seven or above were used. cDNA synthesis and amplification were performed using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Bio, Inc.) using 10 cycles of PCR. cDNA libraries were constructed using the Low Input Library Prep Kit (Takara Bio, Inc.). An Illumina NovaSeq 6000 instrument was used to produce paired-end, 150-nucleotide reads for each RNA sample. Paired-end sequencing of 31 samples produced ∼1.55 billion raw sequencing reads. The Spliced Transcripts Alignment to a Reference (STAR) application (16 (link)) was used to perform sequence alignments to the mm10 (GRCm38) mouse genome reference and GENCODE comprehensive gene annotations (release M17). Overall, 80–88% of the raw sequencing reads were uniquely mapped to genomic sites, resulting in 1.3 billion usable reads. HTSeq was used for counting reads mapped to genomic features (17 (link)), and DESeq2 was used for differential gene expression analysis (18 (link)). P_adj <0.05 cutoff was used to define differentially expressed genes. Gene ontology (GO) analysis of differentially expressed genes was performed using Metascape (19 (link)).

Osipovich A.B., Stancill J.S., Cartailler J.P., Dudek K.D, & Magnuson M.A. (2020). Excitotoxicity and Overnutrition Additively Impair Metabolic Function and Identity of Pancreatic β-Cells. Diabetes, 69(7), 1476-1491.

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Single-cell RNA-seq of mouse DCs

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We sorted 10⁴ DCs as described above into 700 μl trizol. We isolated RNA using RNeasy Micro Kits (Qiagen), and synthesized 0.5–1 ng of RNA into cDNA using the Smart-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Bio). Sequencing libraries were prepared using the Low Input Library Prep Kit (Takara Bio). Libraries were sequenced on an Illumina NextSeq 550 system. Fastqs were aligned to the mm10 reference genome; reads were dereplicated for polymerase chain reaction (PCR) duplicates; and gene counts were generated using STAR v2.5 using ‘–quantMode GeneCounts’. Differential expression analyses were performed with the limma R package.

Maier B., Leader A.M., Chen S.T., Tung N., Chang C., LeBerichel J., Chudnovskiy A., Maskey S., Walker L., Finnigan J.P., Kirkling M.E., Reizis B., Ghosh S., D’Amore N.R., Bhardwaj N., Rothlin C.V., Wolf A., Flores R., Marron T., Rahman A.H., Kenigsberg E., Brown B.D, & Merad M. (2020). A conserved dendritic-cell regulatory program limits antitumour immunity. Nature, 580(7802), 257-262.

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Transcriptome Profiling of FACS-Sorted TRMs

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RNA isolation from 20,000 FACS-sorted TRMs was done with the Qiagen RNeasy micro kit. One nanogram of RNA was synthesized into cDNA using the Smart-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Bio). Sequencing libraries were prepared using the Low Input Library Prep Kit (Takara Bio). Libraries were sequenced on an Illumina NextSeq 550 System. FASTQ files were aligned to the mm10 reference genome, reads were dereplicated for PCR duplicates and gene counts were generated using STAR v.2.5 using quantMode GeneCounts. Differential expression analysis was performed with the limma package in R.

Casanova-Acebes M., Dalla E., Leader A.M., LeBerichel J., Nikolic J., Morales B.M., Brown M., Chang C., Troncoso L., Chen S.T., Sastre-Perona A., Park M.D., Tabachnikova A., Dhainaut M., Hamon P., Maier B., Sawai C.M., Agulló-Pascual E., Schober M., Brown B.D., Reizis B., Marron T., Kenigsberg E., Moussion C., Benaroch P., Aguirre-Ghiso J.A, & Merad M. (2021). Tissue-resident macrophages provide a pro-tumorigenic niche to early NSCLC cells. Nature, 595(7868), 578-584.

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Single-cell RNA-seq Library Preparation

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The SMARTer Ultra Low Input RNA Kit for Sequencing – v3 (Clontech Laboratories, #634851) was used to generate cDNA from a single cell following the manufacturers protocol. The cDNA was then visualized and quantified using the High Sensitivity DNA assay on a 2100 Bioanalyzer (Agilent Technologies, #5067-4626). Sequencing libraries were prepared with 2ng of cDNA using the Low Input Library Prep Kit (Clontech Laboratories, #634947) following the manufacturers protocol. Final libraries were validated via the High Sensitivity DNA assay on a 2100 Bioanalyzer (Agilent Technologies, 5067-4626) and quantified by qPCR using Kapa’s Library Quantification Kit (Kapa Biosystems, #KK4824) on the 7900HT (Applied Biosystems). Libraries were pooled prior to sequencing by 75 bp paired-end reads using reagents from the MiSeq 150 cycle kit v3 (Illumina, #MS-102-3001).

Ho H., Both M.D., Siniard A., Sharma S., Notwell J.H., Wallace M., Leone D.P., Nguyen A., Zhao E., Lee H., Zwilling D., Thompson K.R., Braithwaite S.P., Huentelman M, & Portmann T. (2018). A Guide to Single-Cell Transcriptomics in Adult Rodent Brain: The Medium Spiny Neuron Transcriptome Revisited. Frontiers in Cellular Neuroscience, 12, 159.

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Single-cell RNA-seq of FFPE samples

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Total RNA was extracted from the pooled LCM-captured cells with the NucleoSpin totalRNA FFPE XS kit (Macherey-Nagel, REF740969) with minor modifications. In short, cells in the cap were covered with lysis buffer, incubated at 56°C for 15 min and then spun down. Extracted RNA was bound onto the column with binding buffer and genomic DNA was digested for 15 min in rDNase incubation solution. After two washes, RNA was eluted with 10 μl of RNase-free water. rRNA was removed from the purified RNA with the Ribo-Zero Magnetic Gold Kit (Epicentre, MRZG126). The purified RNA was amplified with the SMARTer Universal Low Input RNA Kit for Sequencing (Clontech, 634938) and libraries were generated with the Low Input Library Prep Kit (Clontech, 634947). Libraries were sequenced on an Illumina HiSeq2000 (100 PE bp) at the Welgene Biotech Company, which generated 6 Gb of data per sample. RNA was quantified and qualitated with the Bioanalyzer 2100 (Agilent Technologies) using a RNA 6000 Pico LabChip (Agilent, 5065–4401). DNA was quantified and qualitated with an Agilent 2200 TapeStation system using High Sensitivity D1000 ScreenTape (Agilent, 5067–5584) and High Sensitivity D1000 Reagents (Agilent, 5067–5585).

Lin C.Y., Huang S.C., Tung C.C., Chou C.H., Gau S.S, & Huang H.S. (2016). Analysis of Genome-Wide Monoallelic Expression Patterns in Three Major Cell Types of Mouse Visual Cortex Using Laser Capture Microdissection. PLoS ONE, 11(9), e0163663.

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Transcriptome Profiling via RNA-seq

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For transcriptome profiling, 1–2 ng of amplified double-stranded cDNA was sheared to 200–500 bp fragments in water using a focused ultrasonicator (Covaris S2 system), using microtubes at a power of ‘10 dc, 5 i, 200 cpb fs' for 40 s. Subsequently the Low Input Library Prep Kit (Clontech) was used for preparation of sequencing libraries. Libraries were validated using the High Sensitivity DNA Kit (Agilent), and sequencing was performed on an Illumina Hiseq 2,000 system. Approximately 30 million 2 × 101 base-pair paired-end reads that passed the Illumina quality control were collected for each sample in four biological replications (Supplementary Table 2).

You Y., Sawikowska A., Neumann M., Posé D., Capovilla G., Langenecker T., Neher R.A., Krajewski P, & Schmid M. (2017). Temporal dynamics of gene expression and histone marks at the Arabidopsis shoot meristem during flowering. Nature Communications, 8, 15120.

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Transcriptome Profiling of FACS-Isolated Cells

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RNA was purified from FACS-isolated cells using PicoPure Kit (Thermo
Fisher Scientific). RNA quality was assessed using RNA 6000 Pico Kit on a 2100
Bioanalyzer (Agilent). Samples with RIN ≥ 7.7 and at least 15 ng of RNA
were used to construct sequencing libraries using Clontech Low Input Library
Prep Kit v2. Libraries were sequenced on a HiSeq 3000, 10 samples per lane, with
single-end 50 bp reads. Raw reads were aligned to the mm10 mouse genome with
annotations provided by UCSC using CobWEB, a proprietary Burrows-Wheeler
Transform method. Reads per kb per million (RPKM) were calculated from aligned
reads using the expectation-maximization algorithm. RPKM was thresholded at 1,
log2 transformed, normalized using the DESeq algorithm and baselined to the
median of all samples. Analyses were performed on transcripts with RPKM >5 in all samples of at least 1 experimental condition (n=17,793 transcripts).
These reasonably expressed transcripts were used in principle-component
analysis. All transcripts with fold change > 3 in at least 1 of the 3
possible pairwise comparisons (n=6,464) were selected, and a 1-way ANOVA was
performed to identify significantly differential genes with FDR-corrected
P<0.05 (n=4,997). Venn diagrams were used to
identify unique and shared gene signatures. Gene sets were submitted to
ToppGene.cchmc.org for identification of pathway and biological process
enrichments.

Schaub J.R., Huppert K.A., Kurial S.N., Hsu B.Y., Cast A.E., Donnelly B., Karns R.A., Chen F., Rezvani M., Luu H.Y., Mattis A.N., Rougemont A.L., Rosenthal P., Huppert S.S, & Willenbring H. (2018). De novo formation of the biliary system by TGFβ-mediated hepatocyte transdifferentiation. Nature, 557(7704), 247-251.

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Total RNA Isolation and Sequencing

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Total RNA was isolated using the RNeasy-Micro Kit (Qiagen) and was processed for deep sequencing using SMARTer Ultra Low Input RNA kit for Sequencing-v3 (Clontech) and Low Input Library Prep Kit (Clontech) following the manufacturer’s protocol. cDNA libraries were sequenced on a HiSeq 2500 sequencer (Illumina).

Miyazaki M., Miyazaki K., Chen K., Jin Y., Turner J., Moore A.J., Saito R., Yoshida K., Ogawa S., Rodewald H.R., Lin Y.C., Kawamoto H, & Murre C. (2017). The E-Id Protein Axis Specifies Innate and Adaptive Lymphoid Cell Fate. Immunity, 46(5), 818-834.e4.

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RNA-seq of nuclear transcriptomes

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RNA-seq was performed in three independent biological replicates for nuc^+XopD and nuc⁻^XopD. For each sample, approximately 500 pg nuclear RNA was extracted from ∼10⁵ nuclei using the RNeasy Plus Micro Kit (Qiagen) and further treated with DNase I (0.05 U per μl, Thermo Fisher Scientific) for 30 min at 37 °C to remove any contaminating genomic DNA. The double-stranded (ds) cDNA was synthesized from ∼500 pg RNA by two rounds of linear amplification using the SMARTer Ultra Low Input RNA for Illumina Sequencing-HV kit (Clontech) according to the manufacturer’s instructions. The concentration and yield of the amplified cDNA was determined using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen). RNA-seq libraries were prepared using the Low Input Library Prep Kit (Clontech) according to the manufacturer’s instructions. The quality and quantity of the RNA-seq libraries was examined using the High Sensitivity DNA Kit (Agilent). Sequencing was performed on NovaSeq 6000 system (Novogene, UK). From 33.2 to 40.9 millions of 2× 150-bp paired-end reads that passed the Illumina quality control filter were collected for each sample (Supplementary Table 8).

You Y., Koczyk G., Nuc M., Morbitzer R., Holmes D.R., von Roepenack-Lahaye E., Hou S., Giudicatti A., Gris C., Manavella P.A., Noël L.D., Krajewski P, & Lahaye T. (2022). The eINTACT system dissects bacterial exploitation of plant osmosignalling to enhance virulence. Nature Plants, 9(1), 128-141.

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Single-cell RNA-seq Library Preparation

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RNA was extracted from single cells and processed for cDNA synthesis using the SMARTer Ultra Low RNA Kit for Illumina Sequencing-HV (Clontech Laboratories). Libraries were prepared using Clontech Low Input Library Prep Kit according to the manufacturer's instructions. An extended protocol can be found in the supplementary material Methods.

Blakeley P., Fogarty N.M., del Valle I., Wamaitha S.E., Hu T.X., Elder K., Snell P., Christie L., Robson P, & Niakan K.K. (2015). Defining the three cell lineages of the human blastocyst by single-cell RNA-seq. Development (Cambridge, England), 142(18), 3151-3165.

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