Library quantification kit

Manufactured by Agilent Technologies

The Library Quantification Kit is a laboratory product designed to accurately measure the concentration of DNA libraries. It provides a reliable and consistent method for quantifying libraries prior to sequencing, ensuring optimal input for downstream applications.

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Lab products found in correlation

2100 bioanalyzer, by Agilent Technologies (2 mentions) 2100 bioanalyzer high sensitivity dna assay, by Agilent Technologies (1 mentions) Nextera xt dna library preparation kit, by Illumina (1 mentions) Hiseq 2000, by Illumina (1 mentions) Rnase free dnase 1, by Illumina (1 mentions) Universal plus mrna seq library prep kit, by Tecan (1 mentions) Agilent 2100 bioanalyzer high sensitivity dna assay, by Agilent Technologies (1 mentions) Tri reagent, by Molecular Research Center (1 mentions) 4200 tapestation and high sensitivity d1k screen tape, by Agilent Technologies (1 mentions) Hiseq 4000, by Illumina (1 mentions) Smart seq v4 ultra low input rna kit, by Takara Bio (1 mentions) Ds 11 spectrophotometer, by DeNovix (1 mentions) 2200 tapestation and high sensitivity d1k screen tape, by Agilent Technologies (1 mentions) Truseq rna sample preparation kit v2, by Illumina (1 mentions) Nanodrop 8000, by Thermo Fisher Scientific (1 mentions) Hiseq 2500, by Illumina (1 mentions) Rneasy kit, by Qiagen (1 mentions) Agilent 2100 bioanalyzer high sensitivity dna chip, by Agilent Technologies (1 mentions)

5 protocols using library quantification kit

Nextera XT DNA Library Preparation

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The cDNA was diluted to ∼300 pg/ul and then processed with the Nextera XT DNA library preparation kit according to the manufacturer’s protocol (Illumina). These libraries were purified and size-selected with an average library size of 300–600 bp as determined by Bioanalyzer 2100 high-sensitivity DNA assay (Agilent).
Functional library concentration was determined with the KAPA Biosystems library quantification kit. The libraries were denatured after quantification and loaded on Illumina HiSeq 2000 for sequencing.

Chaves G., Özel R.E., Rao N.V., Hadiprodjo H., Costa Y.D., Tokuno Z, & Pourmand N. (2017). Metabolic and transcriptomic analysis of Huntington’s disease model reveal changes in intracellular glucose levels and related genes. Heliyon, 3(8), e00381.

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Gene Expression Analysis of Whole Heart Samples

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We collected whole heart specimens from cKO and floxed-control adult animals at 22 weeks of age to include: three male cKO, three male control, three female cKO, and three female control samples. Gene microarray analysis was conducted as previously described (34 (link)). Total RNA was isolated using TRI Reagent (Molecular Research Center, Part # TR118). After isolation, RNA was quantified and treated with RNase free DNase I (Epicentre; Part # D9905K) and repurified using RNA Clean XP magnetic beats (Beckman Coulter Part # A63987). Amplified cDNA libraries were prepared from 200 ng of DNA-free total RNA using the Universal Plus mRNA-Seq Library Prep Kit (NuGEN Technologies, Inc; Part #0509-96). Chip-based capillary electrophoresis on Agilent 2100 Bioanalyzer High Sensitivity DNA assays (Agilent Technologies; Part # 5067-4626) was used to analyze quality of the libraries, which were then quantified with the Takara Library Quantification Kit (Part # 638324).

Cavus O., Williams J., Musa H., El Refaey M., Gratz D., Shaheen R., Schwieterman N.A., Koenig S., Antwi-Boasiako S., Young L.J., Xu X., Han M., Wold L.E., Hund T.J., Mohler P.J, & Bradley E.A. (2021). Giant ankyrin-G regulates cardiac function. The Journal of Biological Chemistry, 296, 100507.

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Single-Cell RNA-seq of Intestinal Stem Cells

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For RNA-seq, between 62,000–210,000 ISCs were sorted directly into Buffer RLT and RNA was isolated using the RNeasy Mikro Kit (Qiagen). QC of samples was done to determine RNA quantity and quality prior to the processing by low input RNA-seq method. The concentration of RNA samples was measured using DS-11 spectrophotometer (DeNovix) and the integrity of RNA was determined by 2100 Bioanalyzer (Agilent Technologies). Approximately few ng of total RNA was used as an input material for the library generation using SMART-seq v4 Ultra Low Input RNA kit (Clontech). Size of the libraries was confirmed using 4200 TapeStation and High Sensitivity D1K screen tape (Agilent Technologies) and their concentration was determined by qPCR based method using Library quantification kit (KAPA). The libraries were multiplexed and then sequenced on Illumina HiSeq4000 (Illumina) to generate >30M of single end 50 base pair reads.

Tauc H.M., Rodriguez-Fernandez I.A., Hackney J.A., Pawlak M., Ronnen Oron T., Korzelius J., Moussa H.F., Chaudhuri S., Modrusan Z., Edgar B.A, & Jasper H. (2021). Age-related changes in polycomb gene regulation disrupt lineage fidelity in intestinal stem cells. eLife, 10, e62250.

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RNAseq Analysis of SUV420H2 Knockdown in PANC-1 Cells

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RNaseq was performed by using PANC-1 cells with the following two knockdown conditions: siNTC (nontargeting control) and siSUV420H2 (using sequence 1; Table S1) in triplicates. Total RNA was extracted by using the Qiagen RNeasy kit per manufacturer’s protocol including the on-column DNase digestion. Quality control of samples was done to determine RNA quantity and quality before their processing by RNaseq. The concentration of total RNA samples was determined by using NanoDrop 8000 (Thermo Fisher). The integrity of RNA samples was determined by using 2100 Bioanalyzer (Agilent Technologies). Approximately 500 ng of total RNA was used as an input for library preparation by using TruSeq RNA Sample Preparation kit v2 (Illumina). The size of the libraries was confirmed by using 2200 TapeStation and High Sensitivity D1K screen tape (Agilent Technologies), and their concentration was determined by the qPCR-based method by using Library quantification kit (KAPA). The libraries were multiplexed and then sequenced on Illumina HiSeq2500 (Illumina) to generate 30M of single-end 100-bp reads.

Viotti M., Wilson C., McCleland M., Koeppen H., Haley B., Jhunjhunwala S., Klijn C., Modrusan Z., Arnott D., Classon M., Stephan J.P, & Mellman I. (2018). SUV420H2 is an epigenetic regulator of epithelial/mesenchymal states in pancreatic cancer. The Journal of Cell Biology, 217(2), 763-777.

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Impact of RNA Integrity on Small RNA Sequencing

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We also explored the effects of RNA integrity on library preparation for small RNA sequencing. To address this issue, we selected peripheral blood samples from 15 healthy volunteers. These samples were collected and processed following the same protocols as previously described, but were selected based on varying RNA integrity number (RIN) values. These values represent the level of RNA degradation in the sample, where 10 and 0 are the highest and lowest quality scores, respectively. The 15 samples were split into 5 groups with average RIN values of 9, 7, 5.4, 2.2 and 0 (Fig. 1c). Small RNA libraries were prepared as previously described, validated and quantified using an Agilent 2100 Bioanalyzer High Sensitivity DNA chip and qRT-PCR with the KAPA library quantification kit.

Lopez J.P., Diallo A., Cruceanu C., Fiori L.M., Laboissiere S., Guillet I., Fontaine J., Ragoussis J., Benes V., Turecki G, & Ernst C. (2015). Biomarker discovery: quantification of microRNAs and other small non-coding RNAs using next generation sequencing. BMC Medical Genomics, 8, 35.

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