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Kapa biosystems kit for library quantitation

Manufactured by Roche
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The Kapa Biosystems kit is a laboratory equipment product designed for library quantitation. It provides the core function of accurately quantifying nucleic acid libraries, which is essential for various applications in molecular biology and genomics research.

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Lab products found in correlation

Lightcycler 480, by Roche (13 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (8 mentions) Truseq library generation kits, by Illumina (7 mentions) Hiseq 2500, by Illumina (5 mentions) Hiseq 2000, by Illumina (4 mentions) Stranded mrna library generation kits, by Agilent Technologies (3 mentions) Nebnext ultra directional rna library prep kit for library generation kit, by Illumina (2 mentions) Rneasy kit, by Qiagen (2 mentions) Nextseq 500, by Illumina (2 mentions) Scriptseq complete kit, by Illumina (1 mentions) Rnaclean xp kit, by Beckman Coulter (1 mentions) Hiseq 2000 flow cell, by Illumina (1 mentions) Bioanalyzer, by Agilent Technologies (1 mentions) Hiseq 2000 system, by Illumina (1 mentions) Hiseq 2500 system, by Illumina (1 mentions) 2100 bioanalyzer, by Agilent Technologies (1 mentions)

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KAPA Biosystems kit (12 citations)

14 protocols using kapa biosystems kit for library quantitation

1

RNA-Seq Library Preparation and Sequencing

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RNA samples were submitted to the Genomics Core Laboratory in the Heflin Center for Genomic Sciences at our institution for sample preparation and sequencing. mRNA‐sequencing was performed on the Illumina HiSeq2000. The quality of the total RNA was assessed using the Agilent 2,100 Bioanalyzer followed by two rounds of poly A+ selection and conversion to cDNA. TruSeq library generation kits were utilized as per the manufacturer's instructions (Illumina, San Diego, CA). The cDNA libraries were quantitated using qPCR in a Roche LightCycler 480 with the Kapa Biosystems kit for library quantitation (Kapa Biosystems, Woburn, MA) prior to cluster generation. Clusters were generated to yield approximately 725–825 K clusters/mm2. Cluster density and quality were determined during the run after the first base addition parameters were assessed. Paired end 2X50bp sequencing runs were performed to align the cDNA sequences to the reference genome.
Amm H.M., DeVilliers P., Srivastava A.R., Diniz M.G., Siegal G.P, & MacDougall M. (2020). Mandibular undifferentiated pleomorphic sarcoma: Molecular analysis of a primary cell population. Clinical and Experimental Dental Research, 6(5), 495-505.
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2

Illumina-based mRNA Sequencing Protocol

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mRNA-sequencing was performed on the Illumina HiSeq2500 using the sequencing reagents and flow cells providing up to 300 Gb of sequence information per flow cell. Briefly, the quality of the total RNA was assessed using the Agilent 2100 Bioanalyzer followed by two rounds of polyA+ selection and conversion to cDNA. We used the stranded mRNA library generation kits per manufacturer's instructions (Agilent, Santa Clara, CA). Library construction consists of random fragmentation of the polyA mRNA, followed by cDNA production using random primers with inclusion of Actinomycin D in the first strand reaction. The ends of the cDNA are repaired, A-tailed and adaptors ligated for indexing (four different barcodes per lane) during the sequencing runs. The cDNA libraries were quantitated using qPCR in a Roche LightCycler 480 with the Kapa Biosystems kit for library quantitation (Kapa Biosystems, Woburn, MA) before cluster generation. Clusters were generated to yield approximately 725 to 825 K clusters per mm2. Cluster density and quality were determined during the run after the first base addition parameters were assessed. We ran paired end 2 × 50 bp sequencing runs to align the cDNA sequences to the reference genome.
Shao Z., Zhang R., Khodadadi-Jamayran A., Chen B., Crowley M.R., Festok M.A., Crossman D.K., Townes T.M, & Hu K. (2016). The acetyllysine reader BRD3R promotes human nuclear reprogramming and regulates mitosis. Nature Communications, 7, 10869.
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3

High-throughput RNA-seq library preparation

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Approximately 500 ng of RNA from each sample was used to prepare individually barcoded strand-specific RNA-seq libraries. rRNA and mitochondrial RNA was removed using Ribo-Zero Gold Removal Magnetic Kit for human/mouse/rat (Epicentre, now Illumina) following the manufacturer’s instructions. mRNA was purified using Agencourt RNAClean XP kit. Libraries were constructed using ScriptSeq™ Complete Kit (Epicentre, now Illumina) as per manufacturer’s instructions. Quality and quantity of the libraries were determined using a Bioanalyzer (Agilent) and Qubit® (Invitrogen), respectively. Libraries were then quantified by qPCR in an Applied Biosystems® 7500 with the Kapa Biosystems kit for library quantitation (Kapa Biosystems). Next-generation sequencing was performed using an Illumina HiSeq 2000 flow cell with one 150-bp end run. The NCBI Gene Expression Omnibus (GEO) accession number for the RNA-seq data reported in this paper is GSE88801.
Andreu N., Phelan J., de Sessions P.F., Cliff J.M., Clark T.G, & Hibberd M.L. (2017). Primary macrophages and J774 cells respond differently to infection with Mycobacterium tuberculosis. Scientific Reports, 7, 42225.
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4

RNA-Seq Workflow with Illumina HiSeq2000

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RNA-sequencing was performed on Illumina HiSeq2000 in UAB Heflin Genomic Core Facility. cDNA libraries were constructed with TruSeq library generation kits (Illumina). The cDNA ends were repaired, A-tailed, and adaptor ligated for indexing. The cDNA library was then quantitated by real time PCR in a Roche LightCycler 480 with the Kapa Biosystems kit for library quantitation (Kapa Biosystems) prior to cluster generation. Clusters were then generated to yield approximately 725–825 K clusters/mm2 and the density and quality were determined after the first base addition. Paired end 2 × 50bp sequencing runs were performed for all samples. After sequencing, the data was converted to FASTQ Sanger format and then aligned to mouse reference genome mm9 by TopHat49 (link). Cufflinks was used to assemble transcripts, estimate the abundances, and test for differential expression. Cuffmerge was used to compare the assembled transcripts to the reference annotation across multiple experiments. Cuffdiff was then used to determine the change in transcript levels. Cufflinks, Cuffmerge and Cuffdiff analyses were performed as described previously50 . Gene ontology analysis was performed using DAVID (http://david.abcc.ncifcrf.gov/).
Yang W., Lee Y.H., Jones A.E., Woolnough J.L., Zhou D., Dai Q., Wu Q., Giles K.E., Townes T.M, & Wang H. (2014). The histone H2A deubiquitinase Usp16 regulates embryonic stem cell gene expression and lineage commitment. Nature communications, 5, 3818.
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5

Illumina HiSeq2500 RNA Sequencing Protocol

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Sequencing of the total RNA was performed at the University of Alabama at Birmingham (UAB) Heflin Center for Genomic Science Core Laboratories. mRNA-sequencing was performed on the Illumina HiSeq2500 using the latest versions of the sequencing reagents and flow cells providing up to 300 Gb of sequence information per flow cell. Briefly, the quality of the total RNA was assessed using the Agilent 2100 Bioanalyzer followed by 2 rounds of poly A+ selection and conversion to cDNA. We used the TruSeq library generation kits as per the manufacturer’s instructions (Illumina, San Diego, CA, USA). Library construction consists of random fragmentation of the polyA mRNA, followed by cDNA production using random primers. The ends of the cDNA were repaired and A-tailed, and adaptors ligated for indexing (up to 12 different barcodes per lane) during the sequencing runs. The cDNA libraries were quantitated using qPCR in a Roche LightCycler 480 with the Kapa Biosystems kit for library quantitation (Kapa Biosystems, Woburn, MA) prior to cluster generation. Clusters were generated to yield approximately 725–825 K clusters/mm2. Cluster density and quality were determined during the run after the first base addition parameters were assessed. We ran paired end 2 × 50 bp sequencing runs to align the cDNA sequences to the reference genome (Table S1).
Hu M., Crossman D., Prasain J.K., Miller M.A, & Serra R.A. (2020). Transcriptomic Profiling of DAF-7/TGFβ Pathway Mutants in C. elegans. Genes, 11(3), 288.
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6

RNA-Seq Workflow with Illumina HiSeq2000

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RNA-sequencing was performed on Illumina HiSeq2000 in UAB Heflin Genomic Core Facility. cDNA libraries were constructed with TruSeq library generation kits (Illumina). The cDNA ends were repaired, A-tailed, and adaptor ligated for indexing. The cDNA library was then quantitated by real time PCR in a Roche LightCycler 480 with the Kapa Biosystems kit for library quantitation (Kapa Biosystems) prior to cluster generation. Clusters were then generated to yield approximately 725–825 K clusters/mm2 and the density and quality were determined after the first base addition. Paired end 2 × 50bp sequencing runs were performed for all samples. After sequencing, the data was converted to FASTQ Sanger format and then aligned to mouse reference genome mm9 by TopHat49 (link). Cufflinks was used to assemble transcripts, estimate the abundances, and test for differential expression. Cuffmerge was used to compare the assembled transcripts to the reference annotation across multiple experiments. Cuffdiff was then used to determine the change in transcript levels. Cufflinks, Cuffmerge and Cuffdiff analyses were performed as described previously50 . Gene ontology analysis was performed using DAVID (http://david.abcc.ncifcrf.gov/).
Yang W., Lee Y.H., Jones A.E., Woolnough J.L., Zhou D., Dai Q., Wu Q., Giles K.E., Townes T.M, & Wang H. (2014). The histone H2A deubiquitinase Usp16 regulates embryonic stem cell gene expression and lineage commitment. Nature communications, 5, 3818.
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7

Transcriptome Profiling via RNA-Seq

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Total cellular RNA was extracted using the RNeasy kit (Qiagen) according to the manufacturer’s protocol. RNA was sent to the UAB Genomics Core for sample quality control (QC), library preparation, and sequencing. Briefly, the quality of the total RNA was assessed using the Agilent 2100 Bioanalyzer followed by 2 rounds of poly A+ selection and conversion to cDNA. The NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® library generation kit (New England Biolabs) was used per the manufacturer’s instructions. The libraries were quantitated using qPCR in a Roche LightCycler 480 with the Kapa Biosystems kit for library quantitation (Kapa Biosystems, Woburn, MA). Subsequently, sequencing was performed on the Illumina NextSeq500 using the latest versions of the sequencing reagents and flow cells with single-end 75 bp reads. Raw and processed data were deposited in Gene Expression Omnibus (GEO, Accession # GSE164082).
Marayati R., Stafman L.L., Williams A.P., Bownes L.V., Quinn C.H., Markert H.R., Easlick J.L., Stewart J.E., Crossman D.K., Mroczek-Musulman E, & Beierle E.A. (2021). CRISPR/Cas9-mediated knockout of PIM3 suppresses tumorigenesis and cancer cell stemness in human hepatoblastoma cells. Cancer gene therapy, 29(5), 558-572.
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8

Illumina HiSeq2500 mRNA Sequencing

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mRNA-sequencing was carried out on the Illumina HiSeq2500 following the established protocols. RNA-seq library preparation was done using the Agilent SureSelect Stranded kit (Agilent, Santa Clara, CA) as per the manufacturer's instruction. The libraries were quantitated using qPCR in a Roche LightCycler 480 with the Kapa Biosystems kit for library quantitation (Kapa Biosystems, Woburn, MA) both immediately prior to and after library construction. We conducted paired end 50-bp sequencing for downstream analyses.
Hu K., Ianov L, & Crossman D. (2020). Profiling and quantification of pluripotency reprogramming reveal that WNT pathways and cell morphology have to be reprogramed extensively. Heliyon, 6(5), e04035.
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9

RNA-Seq Analysis of hns Mutant

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RNA-Seq was performed on the Illumina HiSeq2000 system using the latest versions of sequencing reagents and flow cells providing up to 300 Gb of sequence information per cell. The quality of the RNA was assessed using the Agilent 2100 Bioanalyzer and samples were subsequently converted to cDNA. Libraries were constructed using the TruSeq library generation kits as per the manufacturer’s instructions (Illumina, San Diego, CA). The cDNA libraries were quantitated using qPCR in a Roche LightCycler 480 with the Kapa Biosystems kit for library quantitation (Kapa Biosystems, Woburn, MA) prior to cluster generation. Clusters were generated to yield approximately 725K – 825K clusters/mm2. Paired-end 50 bp sequencing runs were conducted to align the cDNA sequences to the reference genome. The TopHat software and the short read aligner Bowtie were used to align the raw RNA-Seq fastq reads to the reference genome (Tables 1 and 2). Transcripts assembly, abundance and evaluation of differential expression were accomplished using the Cufflinks software. Genes exhibiting a fold change ≥ ±2.0 and q-value < 0.05 were considered differentially expressed in the hns mutant.
Ayala J.C., Wang H., Benitez J.A, & Silva A.J. (2015). RNA-Seq analysis and whole genome DNA-binding profile of the Vibrio cholerae histone-like nucleoid structuring protein (H-NS). Genomics Data, 5, 147-150.
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10

RNA-Seq Workflow for Mouse Transcriptome Analysis

Cited 1 time
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RNA-Seq was performed using 50nt paired-end reads on the Illumina HiSeq 2500 system at the UAB Heflin Center for Genomic Sciences. RNA quality was assessed using an Agilent 2100 Bioanalyzer and samples were subsequently converted to cDNA. Libraries were constructed using the TruSeq library generation kits as per the manufacturer’s instructions (Illumina, San Diego, CA). cDNA libraries were quantitated using qPCR in a Roche LightCycler 480 with the Kapa Biosystems kit for library quantitation (Kapa Biosystems, Woburn, MA) prior to cluster generation. Clusters were generated to yield approximately 725 K–825 K clusters/mm2.
The quality of the sequencing reads was verified with FastQC (version 0.11.3; http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The mean Phred quality score at almost every position was found to be above 30, except in one sample, which was excluded from further analysis. Reads were aligned using Tophat 2.0.10 against the mm10 mouse reference downloaded from Illumina’s iGenome resource (https://support.illumina.com/sequencing/sequencing_software/igenome.html) (60 (link)). MultiQC was used to produce a combined QC report for sequence quality and alignment (61 (link)).
Kahan S.M., Bakshi R.K., Ingram J.T., Hendrickson R.C., Lefkowitz E.J., Crossman D.K., Harrington L.E., Weaver C.T, & Zajac A.J. (2022). Intrinsic IL-2 Production by Effector CD8 T Cells Affects IL-2 Signaling and Promotes Fate Decisions, Stemness, and Protection. Science immunology, 7(68), eabl6322.
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