kapa library quantification kit  (Kapa Biosystems)

 
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  • 99
    Name:
    KAPA Library Quantification kit
    Description:

    Catalog Number:
    KK4824
    Price:
    None
    Score:
    85
    Buy from Supplier


    Structured Review

    Kapa Biosystems kapa library quantification kit
    Schematic of the study. (A) Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) workflow. Following treatment with pulses of gonadotropin-releasing hormone (GnRH), LβT2 cells cultured in coverslips placed in racks [see in Ref. ] were harvested by trypsinization and resuspended in medium, as indicated. Cell samples were lysed, generating crude nuclei preparations. Next, the transposition reaction was performed in the presence of Tn5 transposase, which cuts and ligates adapters at <t>DNA</t> regions of increased accessibility. The quality of the transposed DNA fragment libraries was assessed using a Bioanalyzer. Libraries were sequenced, mapped to the genome, and processed bioinformatically, and accessible genomic regions were detected (peak calling). (B) Single-cell (SC) RNA-seq [gel bead-in-emulsion (GEM) Drop-seq] workflow. Following treatment with GnRH or vehicle, cultured LβT2 cells were harvested by trypsinization and resuspended in a PBS BSA-containing buffer. Cell samples were loaded on a microfluidic chip, combined with reagents and barcoded gel beads to form Gel beads in Emulsion (GEMs), and then mixed with oil. The resulting emulsions were collected and reverse transcribed in SC droplets. Oil was removed, and barcoded cDNA was amplified, purified, and subject to quality control (QC) assessment. Amplified cDNA was incorporated into libraries, which were subsequently subject to QC evaluation using <t>Kapa,</t> Bioanalyzer, and Qubit. Libraries were pooled for sequencing and demultiplexed for subsequent analyses.

    https://www.bioz.com/result/kapa library quantification kit/product/Kapa Biosystems
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    Images

    1) Product Images from "Regulatory Architecture of the LβT2 Gonadotrope Cell Underlying the Response to Gonadotropin-Releasing Hormone"

    Article Title: Regulatory Architecture of the LβT2 Gonadotrope Cell Underlying the Response to Gonadotropin-Releasing Hormone

    Journal: Frontiers in Endocrinology

    doi: 10.3389/fendo.2018.00034

    Schematic of the study. (A) Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) workflow. Following treatment with pulses of gonadotropin-releasing hormone (GnRH), LβT2 cells cultured in coverslips placed in racks [see in Ref. ] were harvested by trypsinization and resuspended in medium, as indicated. Cell samples were lysed, generating crude nuclei preparations. Next, the transposition reaction was performed in the presence of Tn5 transposase, which cuts and ligates adapters at DNA regions of increased accessibility. The quality of the transposed DNA fragment libraries was assessed using a Bioanalyzer. Libraries were sequenced, mapped to the genome, and processed bioinformatically, and accessible genomic regions were detected (peak calling). (B) Single-cell (SC) RNA-seq [gel bead-in-emulsion (GEM) Drop-seq] workflow. Following treatment with GnRH or vehicle, cultured LβT2 cells were harvested by trypsinization and resuspended in a PBS BSA-containing buffer. Cell samples were loaded on a microfluidic chip, combined with reagents and barcoded gel beads to form Gel beads in Emulsion (GEMs), and then mixed with oil. The resulting emulsions were collected and reverse transcribed in SC droplets. Oil was removed, and barcoded cDNA was amplified, purified, and subject to quality control (QC) assessment. Amplified cDNA was incorporated into libraries, which were subsequently subject to QC evaluation using Kapa, Bioanalyzer, and Qubit. Libraries were pooled for sequencing and demultiplexed for subsequent analyses.
    Figure Legend Snippet: Schematic of the study. (A) Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) workflow. Following treatment with pulses of gonadotropin-releasing hormone (GnRH), LβT2 cells cultured in coverslips placed in racks [see in Ref. ] were harvested by trypsinization and resuspended in medium, as indicated. Cell samples were lysed, generating crude nuclei preparations. Next, the transposition reaction was performed in the presence of Tn5 transposase, which cuts and ligates adapters at DNA regions of increased accessibility. The quality of the transposed DNA fragment libraries was assessed using a Bioanalyzer. Libraries were sequenced, mapped to the genome, and processed bioinformatically, and accessible genomic regions were detected (peak calling). (B) Single-cell (SC) RNA-seq [gel bead-in-emulsion (GEM) Drop-seq] workflow. Following treatment with GnRH or vehicle, cultured LβT2 cells were harvested by trypsinization and resuspended in a PBS BSA-containing buffer. Cell samples were loaded on a microfluidic chip, combined with reagents and barcoded gel beads to form Gel beads in Emulsion (GEMs), and then mixed with oil. The resulting emulsions were collected and reverse transcribed in SC droplets. Oil was removed, and barcoded cDNA was amplified, purified, and subject to quality control (QC) assessment. Amplified cDNA was incorporated into libraries, which were subsequently subject to QC evaluation using Kapa, Bioanalyzer, and Qubit. Libraries were pooled for sequencing and demultiplexed for subsequent analyses.

    Techniques Used: Next-Generation Sequencing, Cell Culture, RNA Sequencing Assay, Chromatin Immunoprecipitation, Amplification, Purification, Sequencing

    2) Product Images from "Regulatory Architecture of the LβT2 Gonadotrope Cell Underlying the Response to Gonadotropin-Releasing Hormone"

    Article Title: Regulatory Architecture of the LβT2 Gonadotrope Cell Underlying the Response to Gonadotropin-Releasing Hormone

    Journal: Frontiers in Endocrinology

    doi: 10.3389/fendo.2018.00034

    Schematic of the study. (A) Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) workflow. Following treatment with pulses of gonadotropin-releasing hormone (GnRH), LβT2 cells cultured in coverslips placed in racks [see in Ref. ( 9 )] were harvested by trypsinization and resuspended in medium, as indicated. Cell samples were lysed, generating crude nuclei preparations. Next, the transposition reaction was performed in the presence of Tn5 transposase, which cuts and ligates adapters at DNA regions of increased accessibility. The quality of the transposed DNA fragment libraries was assessed using a Bioanalyzer. Libraries were sequenced, mapped to the genome, and processed bioinformatically, and accessible genomic regions were detected (peak calling). (B) Single-cell (SC) RNA-seq [gel bead-in-emulsion (GEM) Drop-seq] workflow. Following treatment with GnRH or vehicle, cultured LβT2 cells were harvested by trypsinization and resuspended in a PBS BSA-containing buffer. Cell samples were loaded on a microfluidic chip, combined with reagents and barcoded gel beads to form Gel beads in Emulsion (GEMs), and then mixed with oil. The resulting emulsions were collected and reverse transcribed in SC droplets. Oil was removed, and barcoded cDNA was amplified, purified, and subject to quality control (QC) assessment. Amplified cDNA was incorporated into libraries, which were subsequently subject to QC evaluation using Kapa, Bioanalyzer, and Qubit. Libraries were pooled for sequencing and demultiplexed for subsequent analyses.
    Figure Legend Snippet: Schematic of the study. (A) Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) workflow. Following treatment with pulses of gonadotropin-releasing hormone (GnRH), LβT2 cells cultured in coverslips placed in racks [see in Ref. ( 9 )] were harvested by trypsinization and resuspended in medium, as indicated. Cell samples were lysed, generating crude nuclei preparations. Next, the transposition reaction was performed in the presence of Tn5 transposase, which cuts and ligates adapters at DNA regions of increased accessibility. The quality of the transposed DNA fragment libraries was assessed using a Bioanalyzer. Libraries were sequenced, mapped to the genome, and processed bioinformatically, and accessible genomic regions were detected (peak calling). (B) Single-cell (SC) RNA-seq [gel bead-in-emulsion (GEM) Drop-seq] workflow. Following treatment with GnRH or vehicle, cultured LβT2 cells were harvested by trypsinization and resuspended in a PBS BSA-containing buffer. Cell samples were loaded on a microfluidic chip, combined with reagents and barcoded gel beads to form Gel beads in Emulsion (GEMs), and then mixed with oil. The resulting emulsions were collected and reverse transcribed in SC droplets. Oil was removed, and barcoded cDNA was amplified, purified, and subject to quality control (QC) assessment. Amplified cDNA was incorporated into libraries, which were subsequently subject to QC evaluation using Kapa, Bioanalyzer, and Qubit. Libraries were pooled for sequencing and demultiplexed for subsequent analyses.

    Techniques Used: Next-Generation Sequencing, Cell Culture, RNA Sequencing Assay, Chromatin Immunoprecipitation, Amplification, Purification, Sequencing

    Related Articles

    Methylation Sequencing:

    Article Title: C-Src confers resistance to mitotic stress through inhibition DMAP1/Bub3 complex formation in pancreatic cancer
    Article Snippet: Bisulfite sequencing libraries were constructed using the Illumina TruSeq DNA Library Preparation kit protocol. .. To achieve > 10× genome coverage, libraries were quan¬tified by qPCR with the Library Quantification kit for Illumina sequencing platforms (KK4824, Kapa Biosystems), using the 7900HT Real-Time PCR System (Applied Biosystems), and sequenced on the Illumina HiSeq 2000.

    Amplification:

    Article Title: The Effect of Butyrate-Supplemented Parenteral Nutrition on Intestinal Defence Mechanisms and the Parenteral Nutrition-Induced Shift in the Gut Microbiota in the Rat Model
    Article Snippet: After PCR amplification, triplicates were pooled and the amplified PCR products were determined by gel electrophoresis. .. Differently indexed samples were quantified using the KAPA Library Quantification Complete Kit (Kapa Biosystems, USA) and equimolarly pooled according to the measured concentration.

    Article Title: In-solution Y-chromosome capture-enrichment on ancient DNA libraries
    Article Snippet: All four aliquots of each amplified library were combined, and the library was purified with the Qiagen MinElute PCR purification kit following manufacturer’s instructions with the following modification: the EB buffer was preheated to 65 °C before use, and reactions were eluted in 30 μl. .. Purified libraries were further diluted to a factor of 1:1000 and quantified with the KAPA Library Quantification kit (Kapa Biosystems) following manufacturer’s instructions.

    Article Title: Integrated genetic and epigenetic analysis of myxofibrosarcoma
    Article Snippet: Purification was performed with Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA). .. Quantification and size distribution of the amplified libraries were determined using a BioAnalyzer (Agilent Technologies) and KAPA Library Quantification Kit (KAPA Biosystems). .. Paired-end reads were aligned to the human reference genome (GRCh37) using the Burrows–Wheeler Aligner (BWA) for both tumor and non-tumor samples.

    Article Title: Genome-wide mapping of 5-hydroxymethyluracil in the eukaryote parasite Leishmania
    Article Snippet: The combined eluted DNA solutions were purified using the GeneJET PCR purification kit (Thermo Scientific). .. The obtained 5hmU enriched libraries were amplified by PCR Master Mix and PCR Primer Cocktail provided in the TruSeq DNA Sample Preparation Kit v3 (Illumina) and quantified using a KAPA Library Quantification Kit (KAPA Biosystems). .. Experiments were carried out in technical triplicates for L. major and technical duplicates for L. donovani .

    Article Title: Cannabidiol Modulates the Expression of Alzheimer’s Disease-Related Genes in Mesenchymal Stem Cells
    Article Snippet: After, the libraries were purified through the AMPure XP bead, and amplified according to the protocol (10 cycles; incubation at 98 °C for 10 s, incubation at 60 °C for 30 s and incubation at 72 °C for 30 s), followed by a purification step. .. Library quantification was done through Real-Time PCR using the KAPA Library Quantification Kit-Illumina/ABI Prism® (Kapa Biosystems, Inc., Wilmington, MA, USA).

    Article Title: Impaired DNA replication derepresses chromatin and generates a transgenerationally inherited epigenetic memory
    Article Snippet: All purification steps were performed using AMPure XP Beads (reference no. A63880, Beckman Coulter). .. Final libraries were analyzed using an Agilent DNA 1000 chip to estimate the quantity and to check size distribution and were then quantified by qPCR using the KAPA Library Quantification Kit (reference no. KK4835, Kapa Biosystems) before amplification with Illumina’s cBot. .. Indexed libraries were loaded at a concentration of 2 pM onto the flow cell (12 pM per lane) and were sequenced 1 × 50 on Illumina’s HiSeq 2000.

    Article Title: Cell-type-specific brain methylomes profiled via ultralow-input microfluidics
    Article Snippet: Paragraph title: PCR amplification and sequencing of RRBS samples ... Each library was quantified by KAPA library quantification kit (Kapa Biosystems).

    Article Title: A multi-ethnic meta-analysis confirms the association of rs6570507 with adolescent idiopathic scoliosis
    Article Snippet: We amplified 9,847 bp consisting of 47 genomic fractions using three multiplex-PCR products with dual barcodes for each individual. .. After purifying of each library using Agencourt AMPure XP (Beckman Coulter, CA, USA), the library was applied to the bioanalyzer (Agilent Technologies, CA, USA) to check the size distribution and quantified using the KAPA library quantification kit (Kapa Biosystems, MA, USA) on an ABI Prism 7700 sequence detection system (Life Technologies, CA, USA).

    Synthesized:

    Article Title: Impaired DNA replication derepresses chromatin and generates a transgenerationally inherited epigenetic memory
    Article Snippet: Complementary DNA (cDNA) was synthesized using reverse transcriptase (SuperScript II, reference no. 18064-014, Invitrogen) and random primers. .. Final libraries were analyzed using an Agilent DNA 1000 chip to estimate the quantity and to check size distribution and were then quantified by qPCR using the KAPA Library Quantification Kit (reference no. KK4835, Kapa Biosystems) before amplification with Illumina’s cBot.

    Quantitative RT-PCR:

    Article Title: A minimal RNA ligand for potent RIG-I activation in living mice
    Article Snippet: Libraries were prepared using Illumina TruSeq Stranded mRNA sample preparation kits from 500 ng of purified total RNA according to the manufacturer’s protocol. .. The finished dsDNA libraries were quantified by Qubit fluorometer, Agilent 2200 TapeStation, and qRT-PCR using the Kapa Biosystems library quantification kit according to the manufacturer’s protocols. .. Uniquely indexed libraries were pooled in equimolar ratios and sequenced on an Illumina NextSeq 500 sequencer with paired-end 75-bp reads by the Dana-Farber Cancer Institute Molecular Biology Core Facilities.

    Real-time Polymerase Chain Reaction:

    Article Title: Modulation of Aneuploidy in Leishmania donovani during Adaptation to Different In Vitro and In Vivo Environments and Its Impact on Gene Expression
    Article Snippet: Briefly, DNA-sequencing libraries were prepared using different methods, as follows. (i) The Nextera XT DNA library prep kit (Illumina, Inc.) was used with 1 ng of input genomic DNA according to the manufacturer’s instructions, and the resulting library quantified by quantitative PCR (qPCR) using a KAPA library quantification kit optimized for the Roche LightCycler 480 (Kapa Biosystems) on a LightCycler 480 (Roche). .. The final libraries were quantified with the KAPA library quantification kit (Kapa Biosystems).

    Article Title: Characterization of HIV-1 Near Full-Length Proviral Genome Quasispecies from Patients with Undetectable Viral Load Undergoing First-Line HAART Therapy
    Article Snippet: Briefly, after the fragmentation step using transposon technology, libraries were subjected to a PCR where they were tagmentated with Illumina sequencing adaptors and molecular tags (indexes) to identify each sample. .. A purification step was performed to select fragments of ~800 bp and libraries were quantified by qPCR with the KAPA library quantification kit (Kapa Biosystems, Wilmington, MA, USA). .. Individual libraries were then diluted to 4 nM, considering the mean size of each library and the quantification performed, and pooled.

    Article Title: Nucleotide-resolution DNA double-strand breaks mapping by next-generation sequencing
    Article Snippet: For gDNA sequencing, we sheared gDNA with Covaris S220 AFA (Covaris) according to the manufacturer’s instructions prior to Illumina library preparation. .. We assessed library quality and quantity on the 2100 Bioanalyzer (Agilent) using the High Sensitivity DNA Kit (Agilent), and by qPCR with the Kapa Library quantification Kit (KapaBiosystems). .. We generated clusters on the Illumina flow cell using the automatic cBot station and the TruSeq PE Cluster Kit v3-cBot-HS.

    Article Title: Whole genome sequencing of extended-spectrum β-lactamase producing Klebsiella pneumoniae isolated from a patient in Lebanon
    Article Snippet: After ligation of the adaptors, and to reduce the number of fragments smaller than 500 bp, fragments between 500 and 1000 bp were selected using a Pippin Prep™ DNA size selection system (Sage Science). .. The resulting library was quantified by quantitative PCR in triplicate at 1:1000 and using the Kapa library quantification kit (Kapa Biosystems, Woburn, MA) and as recommended by the manufacturer's protocol. .. The resultant library size was assessed using an Agilent Bioanalyzer with the High Sensitivity DNA Kit.

    Article Title: In-solution Y-chromosome capture-enrichment on ancient DNA libraries
    Article Snippet: After qPCR, all libraries were double-indexed as in [ ]. .. Purified libraries were further diluted to a factor of 1:1000 and quantified with the KAPA Library Quantification kit (Kapa Biosystems) following manufacturer’s instructions.

    Article Title: Cannabidiol Modulates the Expression of Alzheimer’s Disease-Related Genes in Mesenchymal Stem Cells
    Article Snippet: After, the libraries were purified through the AMPure XP bead, and amplified according to the protocol (10 cycles; incubation at 98 °C for 10 s, incubation at 60 °C for 30 s and incubation at 72 °C for 30 s), followed by a purification step. .. Library quantification was done through Real-Time PCR using the KAPA Library Quantification Kit-Illumina/ABI Prism® (Kapa Biosystems, Inc., Wilmington, MA, USA). .. Library validation was performed through a Bioanalyzer with the Agilent High Sensitivity DNA Kit (Richardson, TX, USA).

    Article Title: A Guide to Single-Cell Transcriptomics in Adult Rodent Brain: The Medium Spiny Neuron Transcriptome Revisited
    Article Snippet: 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).

    Article Title: C-Src confers resistance to mitotic stress through inhibition DMAP1/Bub3 complex formation in pancreatic cancer
    Article Snippet: Final libraries were run on the 2100 Bioanalyzer (Agilent) using the High-Sensitivity DNA assay for quality control purposes. .. To achieve > 10× genome coverage, libraries were quan¬tified by qPCR with the Library Quantification kit for Illumina sequencing platforms (KK4824, Kapa Biosystems), using the 7900HT Real-Time PCR System (Applied Biosystems), and sequenced on the Illumina HiSeq 2000. .. The sequences alignment and methylation calling were performed with version 2.43 of BSMAP software.

    Article Title: Impaired DNA replication derepresses chromatin and generates a transgenerationally inherited epigenetic memory
    Article Snippet: All purification steps were performed using AMPure XP Beads (reference no. A63880, Beckman Coulter). .. Final libraries were analyzed using an Agilent DNA 1000 chip to estimate the quantity and to check size distribution and were then quantified by qPCR using the KAPA Library Quantification Kit (reference no. KK4835, Kapa Biosystems) before amplification with Illumina’s cBot. .. Indexed libraries were loaded at a concentration of 2 pM onto the flow cell (12 pM per lane) and were sequenced 1 × 50 on Illumina’s HiSeq 2000.

    Incubation:

    Article Title: Cannabidiol Modulates the Expression of Alzheimer’s Disease-Related Genes in Mesenchymal Stem Cells
    Article Snippet: After, the libraries were purified through the AMPure XP bead, and amplified according to the protocol (10 cycles; incubation at 98 °C for 10 s, incubation at 60 °C for 30 s and incubation at 72 °C for 30 s), followed by a purification step. .. Library quantification was done through Real-Time PCR using the KAPA Library Quantification Kit-Illumina/ABI Prism® (Kapa Biosystems, Inc., Wilmington, MA, USA).

    Article Title: Cell-type-specific brain methylomes profiled via ultralow-input microfluidics
    Article Snippet: 30 μl AMPure bead suspension were then added into the supernatant and incubated for 5 min. .. Each library was quantified by KAPA library quantification kit (Kapa Biosystems).

    Modification:

    Article Title: In-solution Y-chromosome capture-enrichment on ancient DNA libraries
    Article Snippet: All four aliquots of each amplified library were combined, and the library was purified with the Qiagen MinElute PCR purification kit following manufacturer’s instructions with the following modification: the EB buffer was preheated to 65 °C before use, and reactions were eluted in 30 μl. .. Purified libraries were further diluted to a factor of 1:1000 and quantified with the KAPA Library Quantification kit (Kapa Biosystems) following manufacturer’s instructions.

    Article Title: Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing
    Article Snippet: Libraries were quantified using the KAPA Library Quantification kit (Kapa Biosystems). .. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit (75 cycles; Illumina) on a NextSeq 500 platform (Illumina).

    Hybridization:

    Article Title: Cannabidiol Modulates the Expression of Alzheimer’s Disease-Related Genes in Mesenchymal Stem Cells
    Article Snippet: Library quantification was done through Real-Time PCR using the KAPA Library Quantification Kit-Illumina/ABI Prism® (Kapa Biosystems, Inc., Wilmington, MA, USA). .. Library quantification was done through Real-Time PCR using the KAPA Library Quantification Kit-Illumina/ABI Prism® (Kapa Biosystems, Inc., Wilmington, MA, USA).

    Flow Cytometry:

    Article Title: Overexpression of Dimethylarginine Dimethylaminohydrolase 1 Attenuates Airway Inflammation in a Mouse Model of Asthma
    Article Snippet: The size of the generated library was validated by Bioanalyzer and the library was quantified using the Kapa Library Quantification kit (Kapa Biosystems, Woburn, MA). .. The size of the generated library was validated by Bioanalyzer and the library was quantified using the Kapa Library Quantification kit (Kapa Biosystems, Woburn, MA).

    Article Title: Cannabidiol Modulates the Expression of Alzheimer’s Disease-Related Genes in Mesenchymal Stem Cells
    Article Snippet: Library quantification was done through Real-Time PCR using the KAPA Library Quantification Kit-Illumina/ABI Prism® (Kapa Biosystems, Inc., Wilmington, MA, USA). .. The single-read sequencing on the MiSeq instrument (Illumina, Inc., San Diego, CA, USA) was set at 150 cycles.

    Ancient DNA Assay:

    Article Title: In-solution Y-chromosome capture-enrichment on ancient DNA libraries
    Article Snippet: Paragraph title: Ancient DNA library preparation ... Purified libraries were further diluted to a factor of 1:1000 and quantified with the KAPA Library Quantification kit (Kapa Biosystems) following manufacturer’s instructions.

    Methylation:

    Article Title: C-Src confers resistance to mitotic stress through inhibition DMAP1/Bub3 complex formation in pancreatic cancer
    Article Snippet: Ligated DNA was bisulfite converted using the EZ DNA Methylation-Gold kit (ZYMO). .. To achieve > 10× genome coverage, libraries were quan¬tified by qPCR with the Library Quantification kit for Illumina sequencing platforms (KK4824, Kapa Biosystems), using the 7900HT Real-Time PCR System (Applied Biosystems), and sequenced on the Illumina HiSeq 2000.

    Generated:

    Article Title: Overexpression of Dimethylarginine Dimethylaminohydrolase 1 Attenuates Airway Inflammation in a Mouse Model of Asthma
    Article Snippet: The products were purified and enriched by PCR to create the final cDNA library targeting mRNAs. .. The size of the generated library was validated by Bioanalyzer and the library was quantified using the Kapa Library Quantification kit (Kapa Biosystems, Woburn, MA). .. Six individually indexed cDNA libraries were equal amount pooled for clustering in cBot system (Illumina, San Diego, CA).

    Article Title: Metabarcoding analysis of strongylid nematode diversity in two sympatric primate species
    Article Snippet: We generated HTS sequencing libraries using a two-step-PCR approach following Fluidigm Access Array primer design. .. We quantified the library using Kapa Library Quantification Kit (Kapa Biosystems) and sequenced using MiSeq Reagent Kit v2 (2 × 250 bp pair end reads) by Illumina MiSeq platform.

    DNA Sequencing:

    Article Title: Modulation of Aneuploidy in Leishmania donovani during Adaptation to Different In Vitro and In Vivo Environments and Its Impact on Gene Expression
    Article Snippet: Briefly, DNA-sequencing libraries were prepared using different methods, as follows. (i) The Nextera XT DNA library prep kit (Illumina, Inc.) was used with 1 ng of input genomic DNA according to the manufacturer’s instructions, and the resulting library quantified by quantitative PCR (qPCR) using a KAPA library quantification kit optimized for the Roche LightCycler 480 (Kapa Biosystems) on a LightCycler 480 (Roche). .. The final libraries were quantified with the KAPA library quantification kit (Kapa Biosystems).

    Polymerase Chain Reaction:

    Article Title: Characterization of HIV-1 Near Full-Length Proviral Genome Quasispecies from Patients with Undetectable Viral Load Undergoing First-Line HAART Therapy
    Article Snippet: Briefly, after the fragmentation step using transposon technology, libraries were subjected to a PCR where they were tagmentated with Illumina sequencing adaptors and molecular tags (indexes) to identify each sample. .. A purification step was performed to select fragments of ~800 bp and libraries were quantified by qPCR with the KAPA library quantification kit (Kapa Biosystems, Wilmington, MA, USA).

    Article Title: Nucleotide-resolution DNA double-strand breaks mapping by next-generation sequencing
    Article Snippet: We purified PCR products of size comprised between 300 and 800 nt in gel, and analyzed them on the 2100 Bioanalyzer (Agilent) prior to sequencing. .. We assessed library quality and quantity on the 2100 Bioanalyzer (Agilent) using the High Sensitivity DNA Kit (Agilent), and by qPCR with the Kapa Library quantification Kit (KapaBiosystems).

    Article Title: Overexpression of Dimethylarginine Dimethylaminohydrolase 1 Attenuates Airway Inflammation in a Mouse Model of Asthma
    Article Snippet: The products were purified and enriched by PCR to create the final cDNA library targeting mRNAs. .. The size of the generated library was validated by Bioanalyzer and the library was quantified using the Kapa Library Quantification kit (Kapa Biosystems, Woburn, MA).

    Article Title: The Effect of Butyrate-Supplemented Parenteral Nutrition on Intestinal Defence Mechanisms and the Parenteral Nutrition-Induced Shift in the Gut Microbiota in the Rat Model
    Article Snippet: Pools were used as a template for the second PCR with Nextera XT indexes (Illumina, USA). .. Differently indexed samples were quantified using the KAPA Library Quantification Complete Kit (Kapa Biosystems, USA) and equimolarly pooled according to the measured concentration.

    Article Title: Integrated genetic and epigenetic analysis of myxofibrosarcoma
    Article Snippet: Post-capture amplification was performed for 10–11 cycles using Herculase II polymerase with SureSelect Indexing Post-Capture PCR primers. .. Quantification and size distribution of the amplified libraries were determined using a BioAnalyzer (Agilent Technologies) and KAPA Library Quantification Kit (KAPA Biosystems).

    Article Title: Metabarcoding analysis of strongylid nematode diversity in two sympatric primate species
    Article Snippet: We performed the first PCR using Kapa 2 G Robust Hot Star polymerase (Kapa Biosystems), under the following conditions: for the first step, 95 °C for 3 min, (95 °C 15 s, 55.5 °C 15 s, 72 °C 15 s) × 30, and 72 °C for 1 min; and for the second step, 95 °C for 3 min, (95 °C 15 s, 55 °C 30 s, 72 °C 30 s) × 16, and 72 °C for 3 min. We included a total of 40 DNA samples, using in parallel: all DNA isolated from feces (n = 20); and DNA isolated from a mixture of larvae from the coprocultures (n = 20) developed from the same fecal sample. .. We quantified the library using Kapa Library Quantification Kit (Kapa Biosystems) and sequenced using MiSeq Reagent Kit v2 (2 × 250 bp pair end reads) by Illumina MiSeq platform.

    Article Title: Genome-wide mapping of 5-hydroxymethyluracil in the eukaryote parasite Leishmania
    Article Snippet: The combined eluted DNA solutions were purified using the GeneJET PCR purification kit (Thermo Scientific). .. The obtained 5hmU enriched libraries were amplified by PCR Master Mix and PCR Primer Cocktail provided in the TruSeq DNA Sample Preparation Kit v3 (Illumina) and quantified using a KAPA Library Quantification Kit (KAPA Biosystems). .. Experiments were carried out in technical triplicates for L. major and technical duplicates for L. donovani .

    Article Title: Impaired DNA replication derepresses chromatin and generates a transgenerationally inherited epigenetic memory
    Article Snippet: Library amplification was performed by PCR using the primer cocktail supplied in the kit. .. Final libraries were analyzed using an Agilent DNA 1000 chip to estimate the quantity and to check size distribution and were then quantified by qPCR using the KAPA Library Quantification Kit (reference no. KK4835, Kapa Biosystems) before amplification with Illumina’s cBot.

    Article Title: Cell-type-specific brain methylomes profiled via ultralow-input microfluidics
    Article Snippet: Paragraph title: PCR amplification and sequencing of RRBS samples ... Each library was quantified by KAPA library quantification kit (Kapa Biosystems).

    Article Title: A multi-ethnic meta-analysis confirms the association of rs6570507 with adolescent idiopathic scoliosis
    Article Snippet: 2.3.4) to obtain 180–300 bp PCR products. .. After purifying of each library using Agencourt AMPure XP (Beckman Coulter, CA, USA), the library was applied to the bioanalyzer (Agilent Technologies, CA, USA) to check the size distribution and quantified using the KAPA library quantification kit (Kapa Biosystems, MA, USA) on an ABI Prism 7700 sequence detection system (Life Technologies, CA, USA).

    Sonication:

    Article Title: Whole genome sequencing of extended-spectrum β-lactamase producing Klebsiella pneumoniae isolated from a patient in Lebanon
    Article Snippet: Bioruptor® NGS was used to sonicate 1 μg of sample DNA (100 μl final sonication volume in TE buffer) for 8 min of 30 s on/off cycles at 4°C. .. The resulting library was quantified by quantitative PCR in triplicate at 1:1000 and using the Kapa library quantification kit (Kapa Biosystems, Woburn, MA) and as recommended by the manufacturer's protocol.

    Binding Assay:

    Article Title: Genome-wide mapping of 5-hydroxymethyluracil in the eukaryote parasite Leishmania
    Article Snippet: The beads were washed five times with the binding buffer 1 (1×, 100 μL). .. The obtained 5hmU enriched libraries were amplified by PCR Master Mix and PCR Primer Cocktail provided in the TruSeq DNA Sample Preparation Kit v3 (Illumina) and quantified using a KAPA Library Quantification Kit (KAPA Biosystems).

    Article Title: Cannabidiol Modulates the Expression of Alzheimer’s Disease-Related Genes in Mesenchymal Stem Cells
    Article Snippet: Another capture round with streptavidin-coated beads was performed, followed by two heated washes to discharge the non-specific binding from the beads. .. Library quantification was done through Real-Time PCR using the KAPA Library Quantification Kit-Illumina/ABI Prism® (Kapa Biosystems, Inc., Wilmington, MA, USA).

    DNA Extraction:

    Article Title: Metabarcoding analysis of strongylid nematode diversity in two sympatric primate species
    Article Snippet: Paragraph title: DNA isolation and sequencing ... We quantified the library using Kapa Library Quantification Kit (Kapa Biosystems) and sequenced using MiSeq Reagent Kit v2 (2 × 250 bp pair end reads) by Illumina MiSeq platform.

    Nucleic Acid Electrophoresis:

    Article Title: The Effect of Butyrate-Supplemented Parenteral Nutrition on Intestinal Defence Mechanisms and the Parenteral Nutrition-Induced Shift in the Gut Microbiota in the Rat Model
    Article Snippet: After PCR amplification, triplicates were pooled and the amplified PCR products were determined by gel electrophoresis. .. Differently indexed samples were quantified using the KAPA Library Quantification Complete Kit (Kapa Biosystems, USA) and equimolarly pooled according to the measured concentration.

    RNA Sequencing Assay:

    Article Title: Overexpression of Dimethylarginine Dimethylaminohydrolase 1 Attenuates Airway Inflammation in a Mouse Model of Asthma
    Article Snippet: Paragraph title: RNA-Seq and Bioinformatics Analysis ... The size of the generated library was validated by Bioanalyzer and the library was quantified using the Kapa Library Quantification kit (Kapa Biosystems, Woburn, MA).

    Article Title: A minimal RNA ligand for potent RIG-I activation in living mice
    Article Snippet: Paragraph title: RNA-seq library preparation and data analysis ... The finished dsDNA libraries were quantified by Qubit fluorometer, Agilent 2200 TapeStation, and qRT-PCR using the Kapa Biosystems library quantification kit according to the manufacturer’s protocols.

    Article Title: A Guide to Single-Cell Transcriptomics in Adult Rodent Brain: The Medium Spiny Neuron Transcriptome Revisited
    Article Snippet: Paragraph title: NGS Library Generation and Single-Cell RNA-Seq ... 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).

    Article Title: Impaired DNA replication derepresses chromatin and generates a transgenerationally inherited epigenetic memory
    Article Snippet: Paragraph title: RNA sequencing ... Final libraries were analyzed using an Agilent DNA 1000 chip to estimate the quantity and to check size distribution and were then quantified by qPCR using the KAPA Library Quantification Kit (reference no. KK4835, Kapa Biosystems) before amplification with Illumina’s cBot.

    Article Title: Population Substructure Has Implications in Validating Next-Generation Cancer Genomics Studies with TCGA
    Article Snippet: RNA preparations with sufficient mass and integrity (RIN > 7.0) were submitted to the Genomics Division of the University of Iowa Institute for Human Genetics for RNA sequencing (RNAseq) [ ]. .. The concentration of the pools were measured using the Illumina Library Quantification Kit (KAPA Biosystems, Wilmington, MA, USA) and sequenced on the Illumina HiSeq 4000 genome sequencer using a 150 bp paired-end SBS chemistry.

    Magnetic Beads:

    Article Title: Overexpression of Dimethylarginine Dimethylaminohydrolase 1 Attenuates Airway Inflammation in a Mouse Model of Asthma
    Article Snippet: The properly sheared cDNA fragments were purified by Agencourt AMPure XP magnetic beads (Beckman Coulter, Brea CA). .. The size of the generated library was validated by Bioanalyzer and the library was quantified using the Kapa Library Quantification kit (Kapa Biosystems, Woburn, MA).

    Article Title: Cannabidiol Modulates the Expression of Alzheimer’s Disease-Related Genes in Mesenchymal Stem Cells
    Article Snippet: In order to remove non-specific binding, we have used magnetic beads coated with streptavidin to capture probes hybridized at target regions. .. Library quantification was done through Real-Time PCR using the KAPA Library Quantification Kit-Illumina/ABI Prism® (Kapa Biosystems, Inc., Wilmington, MA, USA).

    Isolation:

    Article Title: Metabarcoding analysis of strongylid nematode diversity in two sympatric primate species
    Article Snippet: We performed the first PCR using Kapa 2 G Robust Hot Star polymerase (Kapa Biosystems), under the following conditions: for the first step, 95 °C for 3 min, (95 °C 15 s, 55.5 °C 15 s, 72 °C 15 s) × 30, and 72 °C for 1 min; and for the second step, 95 °C for 3 min, (95 °C 15 s, 55 °C 30 s, 72 °C 30 s) × 16, and 72 °C for 3 min. We included a total of 40 DNA samples, using in parallel: all DNA isolated from feces (n = 20); and DNA isolated from a mixture of larvae from the coprocultures (n = 20) developed from the same fecal sample. .. We quantified the library using Kapa Library Quantification Kit (Kapa Biosystems) and sequenced using MiSeq Reagent Kit v2 (2 × 250 bp pair end reads) by Illumina MiSeq platform.

    Article Title: Population Substructure Has Implications in Validating Next-Generation Cancer Genomics Studies with TCGA
    Article Snippet: Total cellular RNA was purified from individual tumors using the mirVANA mRNA isolation kit following manufacturer’s recommendations (Thermo Fisher Scientific, Waltham, MA, USA). .. The concentration of the pools were measured using the Illumina Library Quantification Kit (KAPA Biosystems, Wilmington, MA, USA) and sequenced on the Illumina HiSeq 4000 genome sequencer using a 150 bp paired-end SBS chemistry.

    Multiplex PCR:

    Article Title: A multi-ethnic meta-analysis confirms the association of rs6570507 with adolescent idiopathic scoliosis
    Article Snippet: We amplified 9,847 bp consisting of 47 genomic fractions using three multiplex-PCR products with dual barcodes for each individual. .. After purifying of each library using Agencourt AMPure XP (Beckman Coulter, CA, USA), the library was applied to the bioanalyzer (Agilent Technologies, CA, USA) to check the size distribution and quantified using the KAPA library quantification kit (Kapa Biosystems, MA, USA) on an ABI Prism 7700 sequence detection system (Life Technologies, CA, USA).

    Purification:

    Article Title: Characterization of HIV-1 Near Full-Length Proviral Genome Quasispecies from Patients with Undetectable Viral Load Undergoing First-Line HAART Therapy
    Article Snippet: Briefly, after the fragmentation step using transposon technology, libraries were subjected to a PCR where they were tagmentated with Illumina sequencing adaptors and molecular tags (indexes) to identify each sample. .. A purification step was performed to select fragments of ~800 bp and libraries were quantified by qPCR with the KAPA library quantification kit (Kapa Biosystems, Wilmington, MA, USA). .. Individual libraries were then diluted to 4 nM, considering the mean size of each library and the quantification performed, and pooled.

    Article Title: Nucleotide-resolution DNA double-strand breaks mapping by next-generation sequencing
    Article Snippet: We purified PCR products of size comprised between 300 and 800 nt in gel, and analyzed them on the 2100 Bioanalyzer (Agilent) prior to sequencing. .. We assessed library quality and quantity on the 2100 Bioanalyzer (Agilent) using the High Sensitivity DNA Kit (Agilent), and by qPCR with the Kapa Library quantification Kit (KapaBiosystems).

    Article Title: Overexpression of Dimethylarginine Dimethylaminohydrolase 1 Attenuates Airway Inflammation in a Mouse Model of Asthma
    Article Snippet: The products were purified and enriched by PCR to create the final cDNA library targeting mRNAs. .. The size of the generated library was validated by Bioanalyzer and the library was quantified using the Kapa Library Quantification kit (Kapa Biosystems, Woburn, MA).

    Article Title: In-solution Y-chromosome capture-enrichment on ancient DNA libraries
    Article Snippet: A 1-μl aliquot of each library was used for quantification with the Qubit 2.0 Broad Range assay. .. Purified libraries were further diluted to a factor of 1:1000 and quantified with the KAPA Library Quantification kit (Kapa Biosystems) following manufacturer’s instructions. .. Indexed libraries were amplified a second time to increase the amount of DNA.

    Article Title: A minimal RNA ligand for potent RIG-I activation in living mice
    Article Snippet: Libraries were prepared using Illumina TruSeq Stranded mRNA sample preparation kits from 500 ng of purified total RNA according to the manufacturer’s protocol. .. The finished dsDNA libraries were quantified by Qubit fluorometer, Agilent 2200 TapeStation, and qRT-PCR using the Kapa Biosystems library quantification kit according to the manufacturer’s protocols.

    Article Title: Integrated genetic and epigenetic analysis of myxofibrosarcoma
    Article Snippet: Purification was performed with Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA). .. Quantification and size distribution of the amplified libraries were determined using a BioAnalyzer (Agilent Technologies) and KAPA Library Quantification Kit (KAPA Biosystems).

    Article Title: Genome-wide mapping of 5-hydroxymethyluracil in the eukaryote parasite Leishmania
    Article Snippet: The combined eluted DNA solutions were purified using the GeneJET PCR purification kit (Thermo Scientific). .. The obtained 5hmU enriched libraries were amplified by PCR Master Mix and PCR Primer Cocktail provided in the TruSeq DNA Sample Preparation Kit v3 (Illumina) and quantified using a KAPA Library Quantification Kit (KAPA Biosystems).

    Article Title: Cannabidiol Modulates the Expression of Alzheimer’s Disease-Related Genes in Mesenchymal Stem Cells
    Article Snippet: After, the libraries were purified through the AMPure XP bead, and amplified according to the protocol (10 cycles; incubation at 98 °C for 10 s, incubation at 60 °C for 30 s and incubation at 72 °C for 30 s), followed by a purification step. .. Library quantification was done through Real-Time PCR using the KAPA Library Quantification Kit-Illumina/ABI Prism® (Kapa Biosystems, Inc., Wilmington, MA, USA).

    Article Title: Impaired DNA replication derepresses chromatin and generates a transgenerationally inherited epigenetic memory
    Article Snippet: All purification steps were performed using AMPure XP Beads (reference no. A63880, Beckman Coulter). .. Final libraries were analyzed using an Agilent DNA 1000 chip to estimate the quantity and to check size distribution and were then quantified by qPCR using the KAPA Library Quantification Kit (reference no. KK4835, Kapa Biosystems) before amplification with Illumina’s cBot.

    Article Title: Population Substructure Has Implications in Validating Next-Generation Cancer Genomics Studies with TCGA
    Article Snippet: Paragraph title: 4.2. RNA Purification and Sequencing ... The concentration of the pools were measured using the Illumina Library Quantification Kit (KAPA Biosystems, Wilmington, MA, USA) and sequenced on the Illumina HiSeq 4000 genome sequencer using a 150 bp paired-end SBS chemistry.

    Sequencing:

    Article Title: Modulation of Aneuploidy in Leishmania donovani during Adaptation to Different In Vitro and In Vivo Environments and Its Impact on Gene Expression
    Article Snippet: Paragraph title: Genome and transcriptome sequencing. ... The final libraries were quantified with the KAPA library quantification kit (Kapa Biosystems).

    Article Title: Characterization of HIV-1 Near Full-Length Proviral Genome Quasispecies from Patients with Undetectable Viral Load Undergoing First-Line HAART Therapy
    Article Snippet: Briefly, after the fragmentation step using transposon technology, libraries were subjected to a PCR where they were tagmentated with Illumina sequencing adaptors and molecular tags (indexes) to identify each sample. .. A purification step was performed to select fragments of ~800 bp and libraries were quantified by qPCR with the KAPA library quantification kit (Kapa Biosystems, Wilmington, MA, USA).

    Article Title: Nucleotide-resolution DNA double-strand breaks mapping by next-generation sequencing
    Article Snippet: For gDNA sequencing, we sheared gDNA with Covaris S220 AFA (Covaris) according to the manufacturer’s instructions prior to Illumina library preparation. .. We assessed library quality and quantity on the 2100 Bioanalyzer (Agilent) using the High Sensitivity DNA Kit (Agilent), and by qPCR with the Kapa Library quantification Kit (KapaBiosystems).

    Article Title: Overexpression of Dimethylarginine Dimethylaminohydrolase 1 Attenuates Airway Inflammation in a Mouse Model of Asthma
    Article Snippet: 10 ng of total RNA was converted into cDNA suitable for mRNA sequencing. .. The size of the generated library was validated by Bioanalyzer and the library was quantified using the Kapa Library Quantification kit (Kapa Biosystems, Woburn, MA).

    Article Title: Whole genome sequencing of extended-spectrum β-lactamase producing Klebsiella pneumoniae isolated from a patient in Lebanon
    Article Snippet: Paragraph title: Genome sequencing ... The resulting library was quantified by quantitative PCR in triplicate at 1:1000 and using the Kapa library quantification kit (Kapa Biosystems, Woburn, MA) and as recommended by the manufacturer's protocol.

    Article Title: The Effect of Butyrate-Supplemented Parenteral Nutrition on Intestinal Defence Mechanisms and the Parenteral Nutrition-Induced Shift in the Gut Microbiota in the Rat Model
    Article Snippet: Each inner tag—a unique sequence of 7–9 bp—was designed to differentiate samples into groups. .. Differently indexed samples were quantified using the KAPA Library Quantification Complete Kit (Kapa Biosystems, USA) and equimolarly pooled according to the measured concentration.

    Article Title: Integrated genetic and epigenetic analysis of myxofibrosarcoma
    Article Snippet: Paragraph title: Library preparation for exome sequencing ... Quantification and size distribution of the amplified libraries were determined using a BioAnalyzer (Agilent Technologies) and KAPA Library Quantification Kit (KAPA Biosystems).

    Article Title: Metabarcoding analysis of strongylid nematode diversity in two sympatric primate species
    Article Snippet: Paragraph title: DNA isolation and sequencing ... We quantified the library using Kapa Library Quantification Kit (Kapa Biosystems) and sequenced using MiSeq Reagent Kit v2 (2 × 250 bp pair end reads) by Illumina MiSeq platform.

    Article Title: Genome-wide mapping of 5-hydroxymethyluracil in the eukaryote parasite Leishmania
    Article Snippet: Paragraph title: Chemical 5hmU enrichment sequencing ... The obtained 5hmU enriched libraries were amplified by PCR Master Mix and PCR Primer Cocktail provided in the TruSeq DNA Sample Preparation Kit v3 (Illumina) and quantified using a KAPA Library Quantification Kit (KAPA Biosystems).

    Article Title: Cannabidiol Modulates the Expression of Alzheimer’s Disease-Related Genes in Mesenchymal Stem Cells
    Article Snippet: Paragraph title: 4.5. Preparation of RNA Libraries for Deep Sequencing ... Library quantification was done through Real-Time PCR using the KAPA Library Quantification Kit-Illumina/ABI Prism® (Kapa Biosystems, Inc., Wilmington, MA, USA).

    Article Title: A Guide to Single-Cell Transcriptomics in Adult Rodent Brain: The Medium Spiny Neuron Transcriptome Revisited
    Article Snippet: 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).

    Article Title: C-Src confers resistance to mitotic stress through inhibition DMAP1/Bub3 complex formation in pancreatic cancer
    Article Snippet: Final libraries were run on the 2100 Bioanalyzer (Agilent) using the High-Sensitivity DNA assay for quality control purposes. .. To achieve > 10× genome coverage, libraries were quan¬tified by qPCR with the Library Quantification kit for Illumina sequencing platforms (KK4824, Kapa Biosystems), using the 7900HT Real-Time PCR System (Applied Biosystems), and sequenced on the Illumina HiSeq 2000. .. The sequences alignment and methylation calling were performed with version 2.43 of BSMAP software.

    Article Title: Impaired DNA replication derepresses chromatin and generates a transgenerationally inherited epigenetic memory
    Article Snippet: Final libraries were analyzed using an Agilent DNA 1000 chip to estimate the quantity and to check size distribution and were then quantified by qPCR using the KAPA Library Quantification Kit (reference no. KK4835, Kapa Biosystems) before amplification with Illumina’s cBot. .. Indexed libraries were loaded at a concentration of 2 pM onto the flow cell (12 pM per lane) and were sequenced 1 × 50 on Illumina’s HiSeq 2000.

    Article Title: Cell-type-specific brain methylomes profiled via ultralow-input microfluidics
    Article Snippet: Paragraph title: PCR amplification and sequencing of RRBS samples ... Each library was quantified by KAPA library quantification kit (Kapa Biosystems).

    Article Title: Population Substructure Has Implications in Validating Next-Generation Cancer Genomics Studies with TCGA
    Article Snippet: Paragraph title: 4.2. RNA Purification and Sequencing ... The concentration of the pools were measured using the Illumina Library Quantification Kit (KAPA Biosystems, Wilmington, MA, USA) and sequenced on the Illumina HiSeq 4000 genome sequencer using a 150 bp paired-end SBS chemistry.

    Article Title: A multi-ethnic meta-analysis confirms the association of rs6570507 with adolescent idiopathic scoliosis
    Article Snippet: We amplified 9,847 bp consisting of 47 genomic fractions using three multiplex-PCR products with dual barcodes for each individual. .. After purifying of each library using Agencourt AMPure XP (Beckman Coulter, CA, USA), the library was applied to the bioanalyzer (Agilent Technologies, CA, USA) to check the size distribution and quantified using the KAPA library quantification kit (Kapa Biosystems, MA, USA) on an ABI Prism 7700 sequence detection system (Life Technologies, CA, USA). .. Sequencing was carried out using the HiSeq.

    Article Title: Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing
    Article Snippet: Paragraph title: Indexing droplets (InDrops) sequencing and analysis ... Libraries were quantified using the KAPA Library Quantification kit (Kapa Biosystems).

    Construct:

    Article Title: C-Src confers resistance to mitotic stress through inhibition DMAP1/Bub3 complex formation in pancreatic cancer
    Article Snippet: Following fragmentation, libraries were constructed using the Illumina Paired-End protocol consisting of end repair, “A” base addition and methylated-adaptor ligation. .. To achieve > 10× genome coverage, libraries were quan¬tified by qPCR with the Library Quantification kit for Illumina sequencing platforms (KK4824, Kapa Biosystems), using the 7900HT Real-Time PCR System (Applied Biosystems), and sequenced on the Illumina HiSeq 2000.

    cDNA Library Assay:

    Article Title: Overexpression of Dimethylarginine Dimethylaminohydrolase 1 Attenuates Airway Inflammation in a Mouse Model of Asthma
    Article Snippet: The products were purified and enriched by PCR to create the final cDNA library targeting mRNAs. .. The size of the generated library was validated by Bioanalyzer and the library was quantified using the Kapa Library Quantification kit (Kapa Biosystems, Woburn, MA).

    Article Title: Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing
    Article Snippet: RNA from 1,500 cells of each sample kidney were pooled and subsequently processed together. cDNA products were preamplified to produce a cDNA library compatible with the Illumina Nextseq platform. .. Libraries were quantified using the KAPA Library Quantification kit (Kapa Biosystems).

    Chromatin Immunoprecipitation:

    Article Title: Impaired DNA replication derepresses chromatin and generates a transgenerationally inherited epigenetic memory
    Article Snippet: All purification steps were performed using AMPure XP Beads (reference no. A63880, Beckman Coulter). .. Final libraries were analyzed using an Agilent DNA 1000 chip to estimate the quantity and to check size distribution and were then quantified by qPCR using the KAPA Library Quantification Kit (reference no. KK4835, Kapa Biosystems) before amplification with Illumina’s cBot. .. Indexed libraries were loaded at a concentration of 2 pM onto the flow cell (12 pM per lane) and were sequenced 1 × 50 on Illumina’s HiSeq 2000.

    Plasmid Preparation:

    Article Title: Impaired DNA replication derepresses chromatin and generates a transgenerationally inherited epigenetic memory
    Article Snippet: Final libraries were analyzed using an Agilent DNA 1000 chip to estimate the quantity and to check size distribution and were then quantified by qPCR using the KAPA Library Quantification Kit (reference no. KK4835, Kapa Biosystems) before amplification with Illumina’s cBot. .. Indexed libraries were loaded at a concentration of 2 pM onto the flow cell (12 pM per lane) and were sequenced 1 × 50 on Illumina’s HiSeq 2000.

    Software:

    Article Title: A multi-ethnic meta-analysis confirms the association of rs6570507 with adolescent idiopathic scoliosis
    Article Snippet: Primers were designed using the Primer 3 software (ver. .. After purifying of each library using Agencourt AMPure XP (Beckman Coulter, CA, USA), the library was applied to the bioanalyzer (Agilent Technologies, CA, USA) to check the size distribution and quantified using the KAPA library quantification kit (Kapa Biosystems, MA, USA) on an ABI Prism 7700 sequence detection system (Life Technologies, CA, USA).

    Multiplex Assay:

    Article Title: A multi-ethnic meta-analysis confirms the association of rs6570507 with adolescent idiopathic scoliosis
    Article Snippet: Multiplex PCR-based target sequencing was carried out as previously described . .. After purifying of each library using Agencourt AMPure XP (Beckman Coulter, CA, USA), the library was applied to the bioanalyzer (Agilent Technologies, CA, USA) to check the size distribution and quantified using the KAPA library quantification kit (Kapa Biosystems, MA, USA) on an ABI Prism 7700 sequence detection system (Life Technologies, CA, USA).

    Selection:

    Article Title: Nucleotide-resolution DNA double-strand breaks mapping by next-generation sequencing
    Article Snippet: For BLESS Illumina library preparation, we used the TruSeq DNA sample preparation kit v2 (Illumina) without DNA fragmentation and library size selection. .. We assessed library quality and quantity on the 2100 Bioanalyzer (Agilent) using the High Sensitivity DNA Kit (Agilent), and by qPCR with the Kapa Library quantification Kit (KapaBiosystems).

    Article Title: Whole genome sequencing of extended-spectrum β-lactamase producing Klebsiella pneumoniae isolated from a patient in Lebanon
    Article Snippet: After ligation of the adaptors, and to reduce the number of fragments smaller than 500 bp, fragments between 500 and 1000 bp were selected using a Pippin Prep™ DNA size selection system (Sage Science). .. The resulting library was quantified by quantitative PCR in triplicate at 1:1000 and using the Kapa library quantification kit (Kapa Biosystems, Woburn, MA) and as recommended by the manufacturer's protocol.

    Sample Prep:

    Article Title: Characterization of HIV-1 Near Full-Length Proviral Genome Quasispecies from Patients with Undetectable Viral Load Undergoing First-Line HAART Therapy
    Article Snippet: Libraries were prepared using the Nextera XT DNA Sample Preparation kit (Illumina Inc., SanDiego, CA, USA) according to the manufacturer’s protocol. .. A purification step was performed to select fragments of ~800 bp and libraries were quantified by qPCR with the KAPA library quantification kit (Kapa Biosystems, Wilmington, MA, USA).

    Article Title: Nucleotide-resolution DNA double-strand breaks mapping by next-generation sequencing
    Article Snippet: For BLESS Illumina library preparation, we used the TruSeq DNA sample preparation kit v2 (Illumina) without DNA fragmentation and library size selection. .. We assessed library quality and quantity on the 2100 Bioanalyzer (Agilent) using the High Sensitivity DNA Kit (Agilent), and by qPCR with the Kapa Library quantification Kit (KapaBiosystems).

    Article Title: A minimal RNA ligand for potent RIG-I activation in living mice
    Article Snippet: Libraries were prepared using Illumina TruSeq Stranded mRNA sample preparation kits from 500 ng of purified total RNA according to the manufacturer’s protocol. .. The finished dsDNA libraries were quantified by Qubit fluorometer, Agilent 2200 TapeStation, and qRT-PCR using the Kapa Biosystems library quantification kit according to the manufacturer’s protocols.

    Article Title: Genome-wide mapping of 5-hydroxymethyluracil in the eukaryote parasite Leishmania
    Article Snippet: The combined eluted DNA solutions were purified using the GeneJET PCR purification kit (Thermo Scientific). .. The obtained 5hmU enriched libraries were amplified by PCR Master Mix and PCR Primer Cocktail provided in the TruSeq DNA Sample Preparation Kit v3 (Illumina) and quantified using a KAPA Library Quantification Kit (KAPA Biosystems). .. Experiments were carried out in technical triplicates for L. major and technical duplicates for L. donovani .

    Next-Generation Sequencing:

    Article Title: Characterization of HIV-1 Near Full-Length Proviral Genome Quasispecies from Patients with Undetectable Viral Load Undergoing First-Line HAART Therapy
    Article Snippet: Paragraph title: 2.3. Library Construction and NGS ... A purification step was performed to select fragments of ~800 bp and libraries were quantified by qPCR with the KAPA library quantification kit (Kapa Biosystems, Wilmington, MA, USA).

    Article Title: Nucleotide-resolution DNA double-strand breaks mapping by next-generation sequencing
    Article Snippet: Paragraph title: Next-generation sequencing ... We assessed library quality and quantity on the 2100 Bioanalyzer (Agilent) using the High Sensitivity DNA Kit (Agilent), and by qPCR with the Kapa Library quantification Kit (KapaBiosystems).

    Article Title: Overexpression of Dimethylarginine Dimethylaminohydrolase 1 Attenuates Airway Inflammation in a Mouse Model of Asthma
    Article Snippet: Using the IntegenX PrepX ILM DNA library kit for Illumina and Apollo 324 NGS Library Prep System, 500 ng of purified cDNA fragments were then put through end repair, addition of a single ‘A’ base and ligation of adapters, and indexed individually. .. The size of the generated library was validated by Bioanalyzer and the library was quantified using the Kapa Library Quantification kit (Kapa Biosystems, Woburn, MA).

    Article Title: Whole genome sequencing of extended-spectrum β-lactamase producing Klebsiella pneumoniae isolated from a patient in Lebanon
    Article Snippet: Bioruptor® NGS was used to sonicate 1 μg of sample DNA (100 μl final sonication volume in TE buffer) for 8 min of 30 s on/off cycles at 4°C. .. The resulting library was quantified by quantitative PCR in triplicate at 1:1000 and using the Kapa library quantification kit (Kapa Biosystems, Woburn, MA) and as recommended by the manufacturer's protocol.

    Article Title: A Guide to Single-Cell Transcriptomics in Adult Rodent Brain: The Medium Spiny Neuron Transcriptome Revisited
    Article Snippet: Paragraph title: NGS Library Generation and Single-Cell RNA-Seq ... 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).

    Ligation:

    Article Title: Overexpression of Dimethylarginine Dimethylaminohydrolase 1 Attenuates Airway Inflammation in a Mouse Model of Asthma
    Article Snippet: Using the IntegenX PrepX ILM DNA library kit for Illumina and Apollo 324 NGS Library Prep System, 500 ng of purified cDNA fragments were then put through end repair, addition of a single ‘A’ base and ligation of adapters, and indexed individually. .. The size of the generated library was validated by Bioanalyzer and the library was quantified using the Kapa Library Quantification kit (Kapa Biosystems, Woburn, MA).

    Article Title: Whole genome sequencing of extended-spectrum β-lactamase producing Klebsiella pneumoniae isolated from a patient in Lebanon
    Article Snippet: After ligation of the adaptors, and to reduce the number of fragments smaller than 500 bp, fragments between 500 and 1000 bp were selected using a Pippin Prep™ DNA size selection system (Sage Science). .. The resulting library was quantified by quantitative PCR in triplicate at 1:1000 and using the Kapa library quantification kit (Kapa Biosystems, Woburn, MA) and as recommended by the manufacturer's protocol.

    Article Title: Integrated genetic and epigenetic analysis of myxofibrosarcoma
    Article Snippet: In brief, 1 μg of DNA was fragmented using a Covaris S2 system (Covaris Inc. Woburn, MA, USA) to produce fragments with an average size of 150–200 base pairs, followed by end repair, A-tailing, and ligation of SureSelect adapter oligos using SureSelect XT Reagents Kit (Agilent Technologies). .. Quantification and size distribution of the amplified libraries were determined using a BioAnalyzer (Agilent Technologies) and KAPA Library Quantification Kit (KAPA Biosystems).

    Article Title: C-Src confers resistance to mitotic stress through inhibition DMAP1/Bub3 complex formation in pancreatic cancer
    Article Snippet: Following fragmentation, libraries were constructed using the Illumina Paired-End protocol consisting of end repair, “A” base addition and methylated-adaptor ligation. .. To achieve > 10× genome coverage, libraries were quan¬tified by qPCR with the Library Quantification kit for Illumina sequencing platforms (KK4824, Kapa Biosystems), using the 7900HT Real-Time PCR System (Applied Biosystems), and sequenced on the Illumina HiSeq 2000.

    Article Title: Impaired DNA replication derepresses chromatin and generates a transgenerationally inherited epigenetic memory
    Article Snippet: Double-stranded DNA was further used for library preparation and was subjected to A-tailing and ligation of the barcoded TruSeq adapters. .. Final libraries were analyzed using an Agilent DNA 1000 chip to estimate the quantity and to check size distribution and were then quantified by qPCR using the KAPA Library Quantification Kit (reference no. KK4835, Kapa Biosystems) before amplification with Illumina’s cBot.

    Spectrophotometry:

    Article Title: Population Substructure Has Implications in Validating Next-Generation Cancer Genomics Studies with TCGA
    Article Snippet: RNA concentration and purity of the collected RNAs were assessed using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and a Model 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). .. The concentration of the pools were measured using the Illumina Library Quantification Kit (KAPA Biosystems, Wilmington, MA, USA) and sequenced on the Illumina HiSeq 4000 genome sequencer using a 150 bp paired-end SBS chemistry.

    Concentration Assay:

    Article Title: Overexpression of Dimethylarginine Dimethylaminohydrolase 1 Attenuates Airway Inflammation in a Mouse Model of Asthma
    Article Snippet: The size of the generated library was validated by Bioanalyzer and the library was quantified using the Kapa Library Quantification kit (Kapa Biosystems, Woburn, MA). .. The size of the generated library was validated by Bioanalyzer and the library was quantified using the Kapa Library Quantification kit (Kapa Biosystems, Woburn, MA).

    Article Title: The Effect of Butyrate-Supplemented Parenteral Nutrition on Intestinal Defence Mechanisms and the Parenteral Nutrition-Induced Shift in the Gut Microbiota in the Rat Model
    Article Snippet: Pools were used as a template for the second PCR with Nextera XT indexes (Illumina, USA). .. Differently indexed samples were quantified using the KAPA Library Quantification Complete Kit (Kapa Biosystems, USA) and equimolarly pooled according to the measured concentration. .. The prepared library was checked with the 2100 Bioanalyzer Instrument (Agilent Technologies, USA), with concentrations measured by qPCR shortly prior to sequencing.

    Article Title: Population Substructure Has Implications in Validating Next-Generation Cancer Genomics Studies with TCGA
    Article Snippet: Molar concentrations of the indexed libraries were measured on the Model 2100 Agilent Bioanalyzer and combined equally into pools for sequencing (Agilent, Santa Clara, CA, USA). .. The concentration of the pools were measured using the Illumina Library Quantification Kit (KAPA Biosystems, Wilmington, MA, USA) and sequenced on the Illumina HiSeq 4000 genome sequencer using a 150 bp paired-end SBS chemistry. .. Endometrial and ovarian cancer data were downloaded from The Cancer Genome Atlas (TCGA) from the National Cancer Institute, following TCGA Human Subject Protection and Data Access Policies.

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    Kapa Biosystems library quantification kit illumina universal
    Library preparation for <t>Illumina</t> sequencing. S2 cells expressing the inducible Hsp70 reporter were treated with dsRNA as indicated in the text. Reporter gene transcription was induced with CuSO4 for 1 h, and total RNA was isolated. After barcoded linker addition, PCR was performed as indicated. A size selection (bracket) was performed for products of 200–300 nt, which were subsequently analyzed by Illumina sequencing.
    Library Quantification Kit Illumina Universal, supplied by Kapa Biosystems, used in various techniques. Bioz Stars score: 99/100, based on 86 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Library preparation for Illumina sequencing. S2 cells expressing the inducible Hsp70 reporter were treated with dsRNA as indicated in the text. Reporter gene transcription was induced with CuSO4 for 1 h, and total RNA was isolated. After barcoded linker addition, PCR was performed as indicated. A size selection (bracket) was performed for products of 200–300 nt, which were subsequently analyzed by Illumina sequencing.

    Journal:

    Article Title: Oligoadenylation of 3′ decay intermediates promotes cytoplasmic mRNA degradation in Drosophila cells

    doi: 10.1261/rna.053942.115

    Figure Lengend Snippet: Library preparation for Illumina sequencing. S2 cells expressing the inducible Hsp70 reporter were treated with dsRNA as indicated in the text. Reporter gene transcription was induced with CuSO4 for 1 h, and total RNA was isolated. After barcoded linker addition, PCR was performed as indicated. A size selection (bracket) was performed for products of 200–300 nt, which were subsequently analyzed by Illumina sequencing.

    Article Snippet: The libraries were purified using the Ampure XP protocol (Beckman Coulter) and quantified using the Library Quantification Kit - Illumina/Universal (KAPA Biosystems) according to the manufacturer's instructions.

    Techniques: Sequencing, Expressing, Isolation, Polymerase Chain Reaction, Selection

    Illumina sequencing analysis of nonencoded 3′ nucleotides. Data were obtained by the procedure outlined in . Labels at the bottom of each figure refer to the dsRNA treatment. Luc, luciferase RNA control; not treated, no dsRNA added; luc-ATP, cells treated with luciferase control RNA and adaptor ligation carried out in the absence of ATP (see Materials and Methods); triple, combined knockdown of CG1091, mkg-p , and Trf4-1. ( A ) Read numbers of individual sequencing libraries in the first and ( B ) in the second experiment. In B , samples “Luc r” and “Mtr3 + Trf4-1 r” are technical replicates of the respective preceding sample. ( C ) Summary of nonencoded 3′ nucleotides in the first and ( D ) in the second experiment. In D , two samples are averages of two technical replicates as explained for B . ( E ) Length distributions of oligo(A) tails summarized over the three control libraries of C at all internal positions [i.e., excluding the regular poly(A) tail] and at the 392/393 hot spot. The number of tails consisting of a single A was set to 1. This plot represents the length distribution up to 20 nt. Note that in all other analyses, only extensions up to 6 nt were included as explained in Materials and Methods. ( F ) Sequence composition of the nonencoded 3′ extensions in the luciferase control knockdown experiment represented as sequence logo. For this analysis, tails of between one and six nonencoded nucleotides were used independently of their sequence. Only tails from internal positions were used, i.e., the regular poly(A) tail was excluded from the analysis.

    Journal:

    Article Title: Oligoadenylation of 3′ decay intermediates promotes cytoplasmic mRNA degradation in Drosophila cells

    doi: 10.1261/rna.053942.115

    Figure Lengend Snippet: Illumina sequencing analysis of nonencoded 3′ nucleotides. Data were obtained by the procedure outlined in . Labels at the bottom of each figure refer to the dsRNA treatment. Luc, luciferase RNA control; not treated, no dsRNA added; luc-ATP, cells treated with luciferase control RNA and adaptor ligation carried out in the absence of ATP (see Materials and Methods); triple, combined knockdown of CG1091, mkg-p , and Trf4-1. ( A ) Read numbers of individual sequencing libraries in the first and ( B ) in the second experiment. In B , samples “Luc r” and “Mtr3 + Trf4-1 r” are technical replicates of the respective preceding sample. ( C ) Summary of nonencoded 3′ nucleotides in the first and ( D ) in the second experiment. In D , two samples are averages of two technical replicates as explained for B . ( E ) Length distributions of oligo(A) tails summarized over the three control libraries of C at all internal positions [i.e., excluding the regular poly(A) tail] and at the 392/393 hot spot. The number of tails consisting of a single A was set to 1. This plot represents the length distribution up to 20 nt. Note that in all other analyses, only extensions up to 6 nt were included as explained in Materials and Methods. ( F ) Sequence composition of the nonencoded 3′ extensions in the luciferase control knockdown experiment represented as sequence logo. For this analysis, tails of between one and six nonencoded nucleotides were used independently of their sequence. Only tails from internal positions were used, i.e., the regular poly(A) tail was excluded from the analysis.

    Article Snippet: The libraries were purified using the Ampure XP protocol (Beckman Coulter) and quantified using the Library Quantification Kit - Illumina/Universal (KAPA Biosystems) according to the manufacturer's instructions.

    Techniques: Sequencing, Luciferase, Ligation

    Schematic of the study. (A) Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) workflow. Following treatment with pulses of gonadotropin-releasing hormone (GnRH), LβT2 cells cultured in coverslips placed in racks [see in Ref. ( 9 )] were harvested by trypsinization and resuspended in medium, as indicated. Cell samples were lysed, generating crude nuclei preparations. Next, the transposition reaction was performed in the presence of Tn5 transposase, which cuts and ligates adapters at DNA regions of increased accessibility. The quality of the transposed DNA fragment libraries was assessed using a Bioanalyzer. Libraries were sequenced, mapped to the genome, and processed bioinformatically, and accessible genomic regions were detected (peak calling). (B) Single-cell (SC) RNA-seq [gel bead-in-emulsion (GEM) Drop-seq] workflow. Following treatment with GnRH or vehicle, cultured LβT2 cells were harvested by trypsinization and resuspended in a PBS BSA-containing buffer. Cell samples were loaded on a microfluidic chip, combined with reagents and barcoded gel beads to form Gel beads in Emulsion (GEMs), and then mixed with oil. The resulting emulsions were collected and reverse transcribed in SC droplets. Oil was removed, and barcoded cDNA was amplified, purified, and subject to quality control (QC) assessment. Amplified cDNA was incorporated into libraries, which were subsequently subject to QC evaluation using Kapa, Bioanalyzer, and Qubit. Libraries were pooled for sequencing and demultiplexed for subsequent analyses.

    Journal: Frontiers in Endocrinology

    Article Title: Regulatory Architecture of the LβT2 Gonadotrope Cell Underlying the Response to Gonadotropin-Releasing Hormone

    doi: 10.3389/fendo.2018.00034

    Figure Lengend Snippet: Schematic of the study. (A) Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) workflow. Following treatment with pulses of gonadotropin-releasing hormone (GnRH), LβT2 cells cultured in coverslips placed in racks [see in Ref. ( 9 )] were harvested by trypsinization and resuspended in medium, as indicated. Cell samples were lysed, generating crude nuclei preparations. Next, the transposition reaction was performed in the presence of Tn5 transposase, which cuts and ligates adapters at DNA regions of increased accessibility. The quality of the transposed DNA fragment libraries was assessed using a Bioanalyzer. Libraries were sequenced, mapped to the genome, and processed bioinformatically, and accessible genomic regions were detected (peak calling). (B) Single-cell (SC) RNA-seq [gel bead-in-emulsion (GEM) Drop-seq] workflow. Following treatment with GnRH or vehicle, cultured LβT2 cells were harvested by trypsinization and resuspended in a PBS BSA-containing buffer. Cell samples were loaded on a microfluidic chip, combined with reagents and barcoded gel beads to form Gel beads in Emulsion (GEMs), and then mixed with oil. The resulting emulsions were collected and reverse transcribed in SC droplets. Oil was removed, and barcoded cDNA was amplified, purified, and subject to quality control (QC) assessment. Amplified cDNA was incorporated into libraries, which were subsequently subject to QC evaluation using Kapa, Bioanalyzer, and Qubit. Libraries were pooled for sequencing and demultiplexed for subsequent analyses.

    Article Snippet: Libraries were purified with AMPure beads and then quantified using a KAPA Library Quantification Kit (Kapa Biosystems) and High-Sensitivity DNA Bionalyzer kit (Agilent) and sequenced on an Illumina HiSeq 2500 to > 164M reads with 50 bp read length, paired-end.

    Techniques: Next-Generation Sequencing, Cell Culture, RNA Sequencing Assay, Chromatin Immunoprecipitation, Amplification, Purification, Sequencing

    Schematic of the study. (A) Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) workflow. Following treatment with pulses of gonadotropin-releasing hormone (GnRH), LβT2 cells cultured in coverslips placed in racks [see in Ref. ] were harvested by trypsinization and resuspended in medium, as indicated. Cell samples were lysed, generating crude nuclei preparations. Next, the transposition reaction was performed in the presence of Tn5 transposase, which cuts and ligates adapters at DNA regions of increased accessibility. The quality of the transposed DNA fragment libraries was assessed using a Bioanalyzer. Libraries were sequenced, mapped to the genome, and processed bioinformatically, and accessible genomic regions were detected (peak calling). (B) Single-cell (SC) RNA-seq [gel bead-in-emulsion (GEM) Drop-seq] workflow. Following treatment with GnRH or vehicle, cultured LβT2 cells were harvested by trypsinization and resuspended in a PBS BSA-containing buffer. Cell samples were loaded on a microfluidic chip, combined with reagents and barcoded gel beads to form Gel beads in Emulsion (GEMs), and then mixed with oil. The resulting emulsions were collected and reverse transcribed in SC droplets. Oil was removed, and barcoded cDNA was amplified, purified, and subject to quality control (QC) assessment. Amplified cDNA was incorporated into libraries, which were subsequently subject to QC evaluation using Kapa, Bioanalyzer, and Qubit. Libraries were pooled for sequencing and demultiplexed for subsequent analyses.

    Journal: Frontiers in Endocrinology

    Article Title: Regulatory Architecture of the LβT2 Gonadotrope Cell Underlying the Response to Gonadotropin-Releasing Hormone

    doi: 10.3389/fendo.2018.00034

    Figure Lengend Snippet: Schematic of the study. (A) Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) workflow. Following treatment with pulses of gonadotropin-releasing hormone (GnRH), LβT2 cells cultured in coverslips placed in racks [see in Ref. ] were harvested by trypsinization and resuspended in medium, as indicated. Cell samples were lysed, generating crude nuclei preparations. Next, the transposition reaction was performed in the presence of Tn5 transposase, which cuts and ligates adapters at DNA regions of increased accessibility. The quality of the transposed DNA fragment libraries was assessed using a Bioanalyzer. Libraries were sequenced, mapped to the genome, and processed bioinformatically, and accessible genomic regions were detected (peak calling). (B) Single-cell (SC) RNA-seq [gel bead-in-emulsion (GEM) Drop-seq] workflow. Following treatment with GnRH or vehicle, cultured LβT2 cells were harvested by trypsinization and resuspended in a PBS BSA-containing buffer. Cell samples were loaded on a microfluidic chip, combined with reagents and barcoded gel beads to form Gel beads in Emulsion (GEMs), and then mixed with oil. The resulting emulsions were collected and reverse transcribed in SC droplets. Oil was removed, and barcoded cDNA was amplified, purified, and subject to quality control (QC) assessment. Amplified cDNA was incorporated into libraries, which were subsequently subject to QC evaluation using Kapa, Bioanalyzer, and Qubit. Libraries were pooled for sequencing and demultiplexed for subsequent analyses.

    Article Snippet: Libraries were purified with AMPure beads and then quantified using a KAPA Library Quantification Kit (Kapa Biosystems) and High-Sensitivity DNA Bionalyzer kit (Agilent) and sequenced on an Illumina HiSeq 2500 to > 164M reads with 50 bp read length, paired-end.

    Techniques: Next-Generation Sequencing, Cell Culture, RNA Sequencing Assay, Chromatin Immunoprecipitation, Amplification, Purification, Sequencing

    Comparison of Illumina high-throughput sequencing and Illumina miRNA Bead Arrays v2 performance in thyroid tumor samples. Each dot represents the abundance of one mature miRNA in 1 of 20 samples in HTS (x-axis) and the fluorescent intensity of the corresponding spot on the microarray (y-axis) of all 20 samples. Expression values are based on normalized data, assuming perfect matching of microarray probe sequence and HTS sequence reads. The figure illustrates the saturation of microarray signals compared to the number of HTS reads and a lack of specificity of microarray probe hybridizations by the presence of fluorescent signal for miRNA targets without any HTS reads (x-axis position -∞). Because RPM normalization of low read counts generates visual artifact (column-like data structures between 0 and 5 RPM) we excluded all miRNAs with a very low abundance of one or two reads in individual samples. The correlation coefficient between the microarray and HTS data set is 0.67.

    Journal: BMC Research Notes

    Article Title: Analysis options for high-throughput sequencing in miRNA expression profiling

    doi: 10.1186/1756-0500-7-144

    Figure Lengend Snippet: Comparison of Illumina high-throughput sequencing and Illumina miRNA Bead Arrays v2 performance in thyroid tumor samples. Each dot represents the abundance of one mature miRNA in 1 of 20 samples in HTS (x-axis) and the fluorescent intensity of the corresponding spot on the microarray (y-axis) of all 20 samples. Expression values are based on normalized data, assuming perfect matching of microarray probe sequence and HTS sequence reads. The figure illustrates the saturation of microarray signals compared to the number of HTS reads and a lack of specificity of microarray probe hybridizations by the presence of fluorescent signal for miRNA targets without any HTS reads (x-axis position -∞). Because RPM normalization of low read counts generates visual artifact (column-like data structures between 0 and 5 RPM) we excluded all miRNAs with a very low abundance of one or two reads in individual samples. The correlation coefficient between the microarray and HTS data set is 0.67.

    Article Snippet: The barcoded libraries were size restricted between 140 and 165 bp, purified and quantified using the Library Quantification Kit - Illumina/Universal (KAPA Biosystems) according to the instructions of the manufacturer.

    Techniques: High Throughput Screening Assay, Sequencing, Microarray, Expressing