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Qpcr library quantification kit

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The QPCR Library Quantification Kit is a laboratory instrument used for the quantification of DNA libraries. It provides an accurate measurement of the concentration of DNA fragments within a library, which is essential for various downstream applications in genomics and molecular biology.

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Lab products found in correlation

Hiseq 2500, by Illumina (5 mentions) Bioanalyzer high sensitivity chip, by Agilent Technologies (2 mentions) Concentrator 5 columns, by Zymo Research (2 mentions) Microplex library preparation kit v2, by Diagenode (2 mentions) Superscript 2, by Thermo Fisher Scientific (2 mentions) Kapa hifi hotstart, by Roche (2 mentions) Ampure bead, by Beckman Coulter (2 mentions) Ampure beads, by Roche (2 mentions) Nextera kit, by Illumina (2 mentions) Hiseq 2000, by Illumina (2 mentions) Gel extraction kit, by Qiagen (1 mentions) Nebnext multiplex oligos for kit, by Illumina (1 mentions) Kapa hifi hotstart pcr mix, by Roche (1 mentions) Dneasy blood and tissue kit, by Qiagen (1 mentions) Pcr purification kit, by Qiagen (1 mentions) Hiseq 4000, by Illumina (1 mentions) Nebnext ultra dna library preparation kit, by Illumina (1 mentions) Agencourt ampure xp bead, by Beckman Coulter (1 mentions) Nucleospin gel and pcr clean up, by Macherey-Nagel (1 mentions) Truseq stranded total rna library prep kit with ribo zero human mouse rat, by Illumina (1 mentions)

Spelling variants of this product

Library quantification kit (120 citations) KAPA qPCR Library Quantification Kit (21 citations) QPCR with Kapa library quantification kit (3 citations) QPCR NGS Library Quantification Kit (2 citations) QPCR-based Library Quantification Kit (2 citations)

10 protocols using qpcr library quantification kit

1

ATAC-seq Libraries for Hematopoietic Cells

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Assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) libraries were generated from freshly prepared cells using the protocol by Buenrostro et al.28 (link). For MKs, 105 cells were used with ten amplification cycles. For HSCs, CMPs and MEPs, 104 cells were used with 12 amplification cycles. Libraries were quantified using a quantitative PCR (qPCR) Library Quantification Kit (Kapa Biosystems), pooled and sequenced with a 50 bp single-end protocol on an Illumina Hiseq 2,500.
Petersen R., Lambourne J.J., Javierre B.M., Grassi L., Kreuzhuber R., Ruklisa D., Rosa I.M., Tomé A.R., Elding H., van Geffen J.P., Jiang T., Farrow S., Cairns J., Al-Subaie A.M., Ashford S., Attwood A., Batista J., Bouman H., Burden F., Choudry F.A., Clarke L., Flicek P., Garner S.F., Haimel M., Kempster C., Ladopoulos V., Lenaerts A.S., Materek P.M., McKinney H., Meacham S., Mead D., Nagy M., Penkett C.J., Rendon A., Seyres D., Sun B., Tuna S., van der Weide M.E., Wingett S.W., Martens J.H., Stegle O., Richardson S., Vallier L., Roberts D.J., Freson K., Wernisch L., Stunnenberg H.G., Danesh J., Fraser P., Soranzo N., Butterworth A.S., Heemskerk J.W., Turro E., Spivakov M., Ouwehand W.H., Astle W.J., Downes K., Kostadima M, & Frontini M. (2017). Platelet function is modified by common sequence variation in megakaryocyte super enhancers. Nature Communications, 8, 16058.
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2

Targeted Next-Gen Sequencing Protocol

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gDNA from cells was extracted using DNeasy Blood and Tissue Kit (Qiagen), according to the manufacturer’s protocol. Next generation sequencing libraries were prepared as follows. Briefly, 4–10 μg of input gDNA was amplified by PCR with primers that amplify 150 bp surrounding the sites of interest (Supplementary Table 1b) using KAPA Hifi HotStart PCR Mix (Kapa Biosystems). PCR products were gel purified (Qiagen Gel Extraction kit), and further purified (Qiagen PCR Purification Kit) to eliminate byproducts. Library construction was done with NEBNext Multiplex Oligos for Illumina kit (NEB). 10–25 ng of input DNA was amplified with indexing primers. Samples were then purified and quantified using a qPCR library quantification kit (Kapa Biosystems, KK4824). Samples were then pooled and loaded on an Illumina Miseq (150 bp paired-end run or 150 single-end run) at 4 nM concentrations. Data analysis was performed using CRISPR Genome Analyzer53 (link).
Katrekar D., Moreno A.M., Chen G., Worlikar A, & Mali P. (2018). Oligonucleotide conjugated multi-functional adeno-associated viruses. Scientific Reports, 8, 3589.
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3

DNA Sequencing Protocol with Zymo Columns

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DNA was recovered with Concentrator 5 columns (Zymo) and prepared for sequencing using MicroPlex Library Preparation Kit v2 (C05010012, Diagenode). Libraries analysed using High Sensitivity Bioanalyzer chips (5067–4626, Agilent), quantified using qPCR Library Quantification Kit (KK4824, Kapa Biosystems), pooled and sequenced with a 50-bp single-end protocol on Illumina HiSeq 2500 or Illumina HiSeq 4000.
Seyres D., Cabassi A., Lambourne J.J., Burden F., Farrow S., McKinney H., Batista J., Kempster C., Pietzner M., Slingsby O., Cao T.H., Quinn P.A., Stefanucci L., Sims M.C., Rehnstrom K., Adams C.L., Frary A., Ergüener B., Kreuzhuber R., Mocciaro G., D’Amore S., Koulman A., Grassi L., Griffin J.L., Ng L.L., Park A., Savage D.B., Langenberg C., Bock C., Downes K., Wareham N.J., Allison M., Vacca M., Kirk P.D, & Frontini M. (2022). Transcriptional, epigenetic and metabolic signatures in cardiometabolic syndrome defined by extreme phenotypes. Clinical Epigenetics, 14, 39.
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4

TP53 Gene Sequencing Protocol

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Target region primers (F-CCTGGTCCTCTGACTGCTCT, R-CTGCCCTGGTAGGTTTTCTG) surrounding the TP53 guide RNA (gRNA.140) recognition site were designed using the batch version of Primer3. The genomic locus was amplified with Phusion High-Fidelity DNA Polymerase (NEB) using 100 ng genomic DNA as input and 20-cycles of PCR. Amplicons were purified using Nucleospin Gel and PCR Clean-up (Macherey–Nagel). Sequencing libraries were prepared using the NEBNext Ultra DNA Library Preparation kit for Illumina (cat# E7370), strictly following the manufacturer's instructions. Briefly, 50 ng of input template was end-repaired, tagged with Illumina adaptors and treated with USER enzyme, followed by limited-cycle PCR with indexing primers. After this amplification, DNA was purified with Agencourt AMPure XP beads (Beckman Coulter). Then, barcoded libraries were pooled in equimolar ratios, quantified by a qPCR library quantification kit (KAPA Biosystems) and subjected to massively parallel 150-bp pair-end sequencing using Miseq Instrument with Miseq Reagent 300 Cycles Kit v2 (Illumina).
Senturk S., Shirole N.H., Nowak D.G., Corbo V., Pal D., Vaughan A., Tuveson D.A., Trotman L.C., Kinney J.B, & Sordella R. (2017). Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization. Nature Communications, 8, 14370.
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5

RNA-seq library preparation using Smart-Seq2

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After RNA isolation, RNA-seq libraries were constructed using the Smart-Seq2 protocol from Trombetta and colleagues [46 (link)], with modifications. Briefly, 1 ng high-quality RNA was reverse transcribed using SuperScript II (Life Technologies, 18064–014) with a poly-dT anchored oligonucleotide primer and a template switching oligonucleotide primer that generated homotypic PCR primer binding sites. The cDNA underwent 10 rounds of PCR amplification using KAPA HiFi Hotstart (Kapa Biosystems, KK2601), followed by Ampure bead (Agencourt) cleanup. The quality of the amplified cDNA was tested using qPCR for GAPDH and nucleic acid quantitation. High-quality amplified cDNA (1 ng) was fragmented with the Tn5 transposase from the Illumina Nextera kit (FC-131-1096) to a median size of approximately 500 bp. The fragmented library was amplified with indexed Nextera adapters (FC-131-1002) using 12 rounds of PCR. Final libraries were purified with Ampure beads and quantified using a qPCR Library Quantification Kit (Kapa Biosystems, KK4824). Libraries were pooled for sequencing on an Illumina HiSeq 2500.
Lin K., Bieri G., Gontier G., Müller S., Smith L.K., Snethlage C.E., White CW I.I.I., Maybury-Lewis S.Y, & Villeda S.A. (2021). MHC class I H2-Kb negatively regulates neural progenitor cell proliferation by inhibiting FGFR signaling. PLoS Biology, 19(6), e3001311.
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6

RNA-Seq Library Generation for Transcriptome Analysis

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RNA sequencing (RNA-seq) libraries were generated by the BLUEPRINT Consortium. In brief, RNA was extracted from TRIzol preparations by phase-separation and precipitation. One microgram of DNase-treated RNA was used to generate ribosomal RNA-depleted libraries with a TruSeq Stranded Total RNA Library Prep Kit (with Ribo-Zero Human/Mouse/Rat, RS-122-2201, Illumina). Libraries were quantified using a qPCR Library Quantification Kit (Kapa Biosystems), pooled and sequenced using paired-end 76 bp sequencing on an Illumina Hiseq 2000.
Petersen R., Lambourne J.J., Javierre B.M., Grassi L., Kreuzhuber R., Ruklisa D., Rosa I.M., Tomé A.R., Elding H., van Geffen J.P., Jiang T., Farrow S., Cairns J., Al-Subaie A.M., Ashford S., Attwood A., Batista J., Bouman H., Burden F., Choudry F.A., Clarke L., Flicek P., Garner S.F., Haimel M., Kempster C., Ladopoulos V., Lenaerts A.S., Materek P.M., McKinney H., Meacham S., Mead D., Nagy M., Penkett C.J., Rendon A., Seyres D., Sun B., Tuna S., van der Weide M.E., Wingett S.W., Martens J.H., Stegle O., Richardson S., Vallier L., Roberts D.J., Freson K., Wernisch L., Stunnenberg H.G., Danesh J., Fraser P., Soranzo N., Butterworth A.S., Heemskerk J.W., Turro E., Spivakov M., Ouwehand W.H., Astle W.J., Downes K., Kostadima M, & Frontini M. (2017). Platelet function is modified by common sequence variation in megakaryocyte super enhancers. Nature Communications, 8, 16058.
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7

Automated ChIP-seq Protocol for Histone Marks and Transcription Factors

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Samples were fixed and prepared using the BLUEPRINT Consortium protocol. In brief, cells were fixed with 1% w/v formaldehyde for 10 min and quenched using 125 mM glycine before washing with PBS. Samples were sonicated using a Bioruptor (Diagenode), final SDS concentration of 0.1% w/v for 9 cycles of 30 s ‘on’ and 30 s ‘off’, and immunoprecipitated using an IP-Star Compact Automated System (Diagenode). For H3-specific antibodies the Auto-Histone ChIP-seq kit protein A (Diagenode) and for CTCF antibody the Auto iDeal ChIP-seq Kit for Transcription Factors (Diagenode) were used with Diagenode antibodies listed in Supplementary Table 6.
Immunoprecipitated and input DNA were reverse cross-linked (65 °C for 4 h), treated with RNase and Proteinase K (65 °C for 30 min). DNA was recovered with Concentrator 5 columns (Zymo) and prepared for sequencing using MicroPlex Library Preparation Kit v2 (Diagenode). Libraries analysed using High Sensitivity Bioanalyzer chips (5,067–4,626, Agilent), quantified using qPCR Library Quantification Kit (Kapa Biosystems), pooled and sequenced with a 50 bp single-end protocol on an Illumina Hiseq 2500.
Petersen R., Lambourne J.J., Javierre B.M., Grassi L., Kreuzhuber R., Ruklisa D., Rosa I.M., Tomé A.R., Elding H., van Geffen J.P., Jiang T., Farrow S., Cairns J., Al-Subaie A.M., Ashford S., Attwood A., Batista J., Bouman H., Burden F., Choudry F.A., Clarke L., Flicek P., Garner S.F., Haimel M., Kempster C., Ladopoulos V., Lenaerts A.S., Materek P.M., McKinney H., Meacham S., Mead D., Nagy M., Penkett C.J., Rendon A., Seyres D., Sun B., Tuna S., van der Weide M.E., Wingett S.W., Martens J.H., Stegle O., Richardson S., Vallier L., Roberts D.J., Freson K., Wernisch L., Stunnenberg H.G., Danesh J., Fraser P., Soranzo N., Butterworth A.S., Heemskerk J.W., Turro E., Spivakov M., Ouwehand W.H., Astle W.J., Downes K., Kostadima M, & Frontini M. (2017). Platelet function is modified by common sequence variation in megakaryocyte super enhancers. Nature Communications, 8, 16058.
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8

PCR-free genomic DNA library prep

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To minimize library bias and coverage gaps associated with PCR amplification of high GC or AT-rich regions we have implemented a version of the TruSeq DNA PCR-free kit (E6875–6877B-GSC, New England Biolabs), automated on a Microlab NIMBUS liquid handling robot (Hamilton Robotics). Briefly, 500 ng of genomic DNA was arrayed in a 96-well microtitre plate and subjected to shearing by sonication (Covaris LE220). Sheared DNA was end-repaired and size selected using paramagnetic PCRClean DX beads (C-1003–450, Aline Biosciences) targeting a 300–400 bp fraction. After 3’ A-tailing, full length TruSeq adapters were ligated. Libraries were purified using paramagnetic (Aline Biosciences) beads. PCR-free genome library concentrations were quantified using a qPCR Library Quantification kit (KAPA, KK4824) prior to sequencing with paired-end 150 base reads on the Illumina HiSeqX platform using V4 chemistry according to manufacturer recommendations.
Michealraj K.A., Kumar S.A., Kim L.J., Cavalli F.M., Przelicki D., Wojcik J.B., Delaidelli A., Bajic A., Saulnier O., MacLeod G., Vellanki R.N., Vladoiu M.C., Guilhamon P., Ong W., Lee J.J., Jiang Y., Holgado B.L., Rasnitsyn A., Malik A.A., Tsai R., Richman C.M., Juraschka K., Haapasalo J., Wang E.Y., De Antonellis P., Suzuki H., Farooq H., Balin P., Kharas K., Van Ommeren R., Sirbu O., Rastan A., Krumholtz S.L., Ly M., Ahmadi M., Deblois G., Srikanthan D., Luu B., Loukides J., Wu X., Garzia L., Ramaswamy V., Kanshin E., Osuna M.S., El-Hamamy I., Coutinho F.J., Prinos P., Singh S., Donovan L.K., Daniels C., Schramek D., Tyers M., Weiss S., Stein L.D., Lupien M., Wouters B.G., Garcia B.A., Arrowsmith C.H., Sorensen P.H., Angers S., Jabado N., Dirks P.B., Mack S.C., Agnihotri S., Rich J.N, & Taylor M.D. (2020). Metabolic Regulation of the Epigenome Drives Lethal Infantile Ependymoma. Cell, 181(6), 1329-1345.e24.
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9

Smart-Seq2 RNA-seq Library Preparation

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After RNA isolation, RNA-sequencing libraries were constructed using the Smart-Seq2 protocol from (46 (link)), with modifications. Briefly, 1 ng high-quality RNA was reverse transcribed using SuperScript II (Life Technologies, 18064-014) with a poly-dT anchored oligonucleotide primer, and a template switching oligonucleotide primer that generated homotypic PCR primer binding sites. The cDNA underwent 10 rounds of PCR amplification using KAPA HiFi Hotstart (Kapa Biosystems, KK2601), followed by Ampure bead (Agencourt) clean-up. The quality of the amplified cDNA was tested using qPCR for GAPDH and nucleic acid quantitation. One nanogram of high-quality amplified cDNA was fragmented with the Tn5 transposase from the Illumina Nextera kit (FC-131-1096) to a median size of ∼500 bp. The fragmented library was amplified with indexed Nextera adapters (FC-131-1002) using 12 rounds of PCR. Final libraries were purified with Ampure beads and quantified using a qPCR Library Quantification Kit (Kapa Biosystems, KK4824). Libraries were pooled for sequencing on an Illumina HiSEq. 2500 (paired reads 2 × 100 bp).
White CW I.I.I., Fan X., Maynard J.C., Wheatley E.G., Bieri G., Couthouis J., Burlingame A.L, & Villeda S.A. (2020). Age-related loss of neural stem cell O-GlcNAc promotes a glial fate switch through STAT3 activation. Proceedings of the National Academy of Sciences of the United States of America, 117(36), 22214-22224.
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10

RNA-Seq Library Preparation and Sequencing

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RNA isolated from samples prepared by different procedures described above was used for total RNA Illumina library preparation using NEB NEXT Ultra RNA library kit (NEB, Ipswich, MA). The quality of libraries was assessed using the qPCR Library Quantification Kit (Kapa Biosystems, Inc., Woburn, MA) and Agilent 2100
Bioanalyzer (Agilent Technologies, Santa Clara, CA). DNA sequencing was performed on Illumina HiSeq 2000 or MiSeq to generate 100 to 300 nt long paired-end reads. In some experiments, samples were sequenced in multiplex format (up to eight samples per sequencing lane) using unique index primers (barcodes).
Furtak V., Roivainen M., Mirochnichenko O., Zagorodnyaya T., Laassri M., Zaidi S.Z., Rehman L., Alam M.M., Chizhikov V, & Chumakov K. (2016). Environmental surveillance of viruses by tangential flow filtration and metagenomic reconstruction. Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin, 21(15).
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