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Nextera sample preparation kit

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The Nextera Sample Preparation Kit is a library preparation solution designed for next-generation sequencing (NGS) workflows. The kit provides a streamlined process for preparing DNA samples, enabling efficient library construction for downstream sequencing analysis.

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Lab products found in correlation

Hiseq 1500 platform, by Illumina (5 mentions) Nextera index kit, by Illumina (5 mentions) Ampure xp bead, by Beckman Coulter (5 mentions) Minelute kit, by Qiagen (4 mentions) Kapa hifi hotstart readymix, by Roche (3 mentions) Minelute pcr purification kit, by Qiagen (3 mentions) Qiaamp dna stool mini kit, by Qiagen (2 mentions) Pcr cleanup kit, by Qiagen (2 mentions) Hiseq 2500 dna sequencing system, by Illumina (2 mentions) Kapa qpcr library quantification kit, by Roche (2 mentions) Picogreen assay, by Thermo Fisher Scientific (2 mentions) 2100 bioanalyzer, by Illumina (1 mentions) Nextseq 500, by Illumina (1 mentions) Agilent 2100 bioanalyzer high sensitivity dna chip, by Agilent Technologies (1 mentions) Quantifluor dye, by Promega (1 mentions) Hiseq 1000 sequencer, by Illumina (1 mentions) Hiseq 2500 sequencer, by Illumina (1 mentions) Dna clean concentrator 5 kit, by Zymo Research (1 mentions) Nextseq, by Illumina (1 mentions) Qubit fluorometer, by Illumina (1 mentions)

18 protocols using nextera sample preparation kit

1

ATAC-Seq Protocol with Modifications

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ATAC-seq was performed as previously described (Buenrostro et al., 2013 (link)) with some modifications. After collecting the GFP-positive compartments into a 1.5 ml tube with PBS, the nuclei were extracted in 500 µl of cold lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Igepal CA-630), centrifuged for 10 min at 500 × g, and supernatant was removed. Tagmentation reaction was performed as described previously (Buenrostro et al., 2013 (link)) with Nextera Sample Preparation Kit (Illumina). After tagmented DNA was purified using MinElute kit (QIAGEN), two sequential PCR were performed to enrich small DNA fragments. First, nine-cycle PCRs were performed using indexed primers from Nextera Index Kit (Illumina) and KAPA HiFi HotStart ReadyMix (KAPA Biosystems), and amplified DNA was size selected to a size of less than 500 bp using AMPure XP beads (Beckman Coulter). Then a second seven-cycle PCR was performed using the same primer as the first PCR and purified by AMPure XP beads. Libraries were sequenced using the Illumina HiSeq 1500 platform.
Isoe Y., Nakamura R., Nonaka S., Kamei Y., Okuyama T., Yamamoto N., Takeuchi H, & Takeda H. (2023). Epigenetically distinct synaptic architecture in clonal compartments in the teleostean dorsal pallium. eLife, 12, e85093.
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2

ATAC-seq on Hepatocytes

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ATAC-seq was performed as described in Buenrostro et al. 2013, with some modifications [82 ]. 50,000 of the sorted hepatocytes were washed with PBS and resuspended with ice-cold lysis buffer (10 mM Tris–HCl, pH 7.4; 10 mM NaCl; 3 mM MgCl2; 0.1% (v/v) Igepal CA-630) to extract nuclei. After centrifugation at 500 × g, 4℃ for 10 min, nuclei pellet was resuspended with the transposition reaction mix (25 µl of 2x reaction buffer and 2.5 µl of Tn5 transposase from Nextera Sample Preparation Kit (Illumina), mixed with 22.5 µl nuclease-free water) and incubated at 37℃ for 30 min. Following transposition, DNA was purified using a Qiagen MinElute PCR Purification Kit (Qiagen) and eluted with 10 µl of Buffer EB. Two sequential PCRs were performed to enrich small DNA fragments. First, nine-cycles of PCR were performed using indexed primers from a Nextera Index kit (Illumina), and amplified DNA was size-selected to a size of < 500 bp using AMPure XP beads (Beckman Coulter). Then, seven cycles of PCR were performed using the same primers as the first PCR and purified by AMPure XP beads. Libraries were sequenced using the Illumina HiSeq 1500 platform.
Inoue Y., Suzuki Y., Kunishima Y., Washio T., Morishita S, & Takeda H. (2023). High-fat diet in early life triggers both reversible and persistent epigenetic changes in the medaka fish (Oryzias latipes). BMC Genomics, 24, 472.
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3

Total RNA Extraction and NGS Library Prep

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Total RNA was extracted using TRIZOL reagent as per manufacturer’s instruction. cDNA libraries were prepared by Nextera sample preparation kit (Illumina, USA). Index labeled libraries sized at 300–450 bp length were recovered by using Ampure XP Beads (Beckman Coulter, USA). All libraries were quantified using 2100 Bioanalyzer and pooled as 1:1 at 2 nM for 150 paired-end modes and were further sequenced by NextSeq 500 (Illumina, USA).
Zhang G., Yan G., Fu Z., wu Y., Wu F., Zheng Z., Fang S., Gao Y., Bao X., Liu Y., Wang X, & Zhu S. (2021). Loss of retinoic acid receptor-related receptor alpha (Rorα) promotes the progression of UV-induced cSCC. Cell Death & Disease, 12(3), 247.
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4

Metagenomic DNA Sequencing for Microbial Diversity

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Metagenomic DNA was quantified using the QuantiFluor dye (Promega). Sequencing libraries were prepared using the Nextera Sample Preparation Kit (Illumina, San Diego, CA, USA) with 50 ng of purified DNA. Dual indices were added during library preparation. Libraries were validated using Agilent Bioanalyzer 2100 high sensitivity DNA chips (Agilent Technologies, Santa Clara, CA, USA) and were quantified again with QuantiFluor. Libraries were diluted to a 2 nm concentration prior to pooling. Two libraries were pooled per HiSeq lane. Sequencing was performed on the HiSeq 1000 sequencer (Illumina) using v3 chemistry and paired-end 101 bp reads. A total of 72 metagenomes were sequenced with an average of 15 Gb per sample (Supplementary Table 1). Metagenomic reads and assemblies have been deposited in the European Nucleotide Archive database in project PRJEB8094.
Raymond F., Ouameur A.A., Déraspe M., Iqbal N., Gingras H., Dridi B., Leprohon P., Plante P.L., Giroux R., Bérubé È., Frenette J., Boudreau D.K., Simard J.L., Chabot I., Domingo M.C., Trottier S., Boissinot M., Huletsky A., Roy P.H., Ouellette M., Bergeron M.G, & Corbeil J. (2015). The initial state of the human gut microbiome determines its reshaping by antibiotics. The ISME Journal, 10(3), 707-720.
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5

Genomic Analysis of Bradyrhizobium Strains

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The type strain B. ottawaense OO99T was purchased from the Microbial Domain Biological Resource Centre HAMBI (Helsinki, Finland). The type strain B. diazoefficiens USDA110T was provided by Dr. Michael J. Sadowsky at University of Minnesota. We used the culture stock of the nosZ deleted mutant B. diazoefficiens USDA110ΔnosZ generated by Hirayama et al. 201151 (link). The B. ottawaense strains used in this study are listed in Supplementary Table 1. Eight strains (SG09, SG10, SG20, SG23, TM102, TM233, and TM239) have been reported by Wasai-Hara et al.17 (link), and the other strains were isolated by the same procedures. For whole genome sequencing, genomic DNA was extracted using a Bacteria GenomicPrep Mini Spin Kit (Cytiva, Tokyo, Japan). DNA libraries were prepared using a Nextera Sample Preparation Kit (Illumina, San Diego, CA, USA), and the 300-bp paired-end libraries were sequenced using Illumina Miseq (Illumina). Subsequently, 20 bp of the 5′ and 3′ ends were trimmed, and the genomes were assembled using CLC Genomics Workbench ver. 8.5.1 (Illumina). Genome annotation was performed using DFAST52 (link).
Wasai-Hara S., Itakura M., Fernandes Siqueira A., Takemoto D., Sugawara M., Mitsui H., Sato S., Inagaki N., Yamazaki T., Imaizumi-Anraku H., Shimoda Y, & Minamisawa K. (2023). Bradyrhizobium ottawaense efficiently reduces nitrous oxide through high nosZ gene expression. Scientific Reports, 13, 18862.
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6

ATAC-seq Library Preparation Protocol

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ATAC-seq protocol was developed by (Buenrostro et al., 2013 (link)). In this study ATAC-seq was performed according to the published protocol with modifications described by (Lara-Astiaso et al., 2014) with a following change; the fragmented DNAs were amplified with total 11 cycles using 1× NEBnext PCR master mix (NEB). In brief, sorted cells were pelleted and washed once with 1x PBS. The cells were pelleted again and were re-suspended in 25 μl of lysis buffer and nuclei were pelleted. The nuclei were re-suspended in 25 μl reaction buffer containing 2 μl of Tn5 transposase and 12.5 μl of TD buffer in the Nextera Sample preparation kit (Illumina) and incubated at 37°C for one hour. Then 5 μl of clean up buffer (900mM NaCl, 300mM EDTA), 2 μl of 5% SDS and 2 μl of Proteinase K (NEB) were added and incubated for 30 min. Tagmentated DNA was isolated using a DNA Clean & Concentrator™-5 kit (ZYMO RESEARCH). For library amplification, two sequential PCR were preformed using 1× NEBnext PCR master mix (NEB). After the first 9-cycle PCR, the libraries were selected for small fragments (less than 600 bp) using SPRI beads. Once the libraries were purified using a DNA Clean & Concentrator™-5 kit (ZYMO RESEARCH), they were sequenced on a HiSeq 2500 sequencer (Illumina).
Miyazaki M., Miyazaki K., Chen K., Jin Y., Turner J., Moore A.J., Saito R., Yoshida K., Ogawa S., Rodewald H.R., Lin Y.C., Kawamoto H, & Murre C. (2017). The E-Id Protein Axis Specifies Innate and Adaptive Lymphoid Cell Fate. Immunity, 46(5), 818-834.e4.
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7

Genomic Analysis of Salmonella Typhimurium

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All isolates were prepared using the Nextera Sample Preparation Kit (Illumina, San Diego, CA) and then sequenced on Illumina NextSeq (Illumina) for 2 x 151 cycles. De novo assemblies were generated from all raw sequence data. The Illumina reads were assembled with SPAdes 3.0 with the following parameters: only contigs of length ≥500 bp were included; mismatch (MM) 3.28; the genome fraction was 96.157; and number of mis-assemblies (MA) was 2 [30 (link)]. The contigs for each isolate (draft genome) were annotated using NCBI’s Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) [31 (link)]. The draft genome sequences of S. Typhimurium strains are publicly available in GenBank, with accession numbers listed in S1 Table. The presence of resistance genes, as well as points mutation in the QRDR of the gyrA, gyrB, parC, and parE genes, were determined using ResFinder (Center for Genomic Epidemiology, https://cge.cbs.dtu.dk/services/ResFinder/) with settings of threshold of 90%, and minimum length of 60% [32 (link)].
Almeida F., Seribelli A.A., Medeiros M.I., Rodrigues D.D., de MelloVarani A., Luo Y., Allard M.W, & Falcão J.P. (2018). Phylogenetic and antimicrobial resistance gene analysis of Salmonella Typhimurium strains isolated in Brazil by whole genome sequencing. PLoS ONE, 13(8), e0201882.
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8

Gut Microbiome and Immune Response Profiling

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Fecal samples were collected on day 4 and day 9 and were analysed by qPCR and 16S rRNA gene-based metagenomic analysis. Blood samples and spleen biopsies were taken on day 9 for immunological studies.
DNA from feces was isolated using QIAamp DNA Stool Mini Kit (Qiagen) following the manufacturer's protocol. Samples were incubated in the lysis buffer at 90 °C for 10 m for optimal bacterial lysis. For microbiome sequencing, DNA libraries were prepared using the Illumina Nextera sample preparation kit with DNA primers corresponding to V3–V4 regions of the 16S rRNA gene listed in Table S1. Illumina MiSeq was used for sequencing the libraries. Sequencing was performed in Saint-Petersburg University Resource Center ≪ Biobank ≫.
Suvorov A., Karaseva A., Kotyleva M., Kondratenko Y., Lavrenova N., Korobeynikov A., Kozyrev P., Kramskaya T., Leontieva G., Kudryavtsev I., Guo D., Lapidus A, & Ermolenko E. (2018). Autoprobiotics as an Approach for Restoration of Personalised Microbiota. Frontiers in Microbiology, 9, 1869.
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9

ATAC-seq of Somite Cells

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ATAC-seq was performed as previously described (Buenrostro et al., 2013 (link)) with some modifications. Approximately 4000 sorted somite cells were used for each experiment. After washing with PBS, cells were resuspended in 500 μl cold lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Igepal CA-630), centrifuged for 10 min at 500 g, supernatant was removed. Tagmentation reaction was performed as described previously (Buenrostro et al., 2013 (link)) with Nextera Sample Preparation Kit (Illumina). After tagmented DNA was purified using MinElute kit (Qiagen), two sequential PCRs were performed to enrich small DNA fragments. First, a 9-cycle PCR was performed using indexed primers from Nextera Index Kit (Illumina) and KAPA HiFi HotStart ReadyMix (KAPA Biosystems), amplified DNA was size selected for a size less than 500 bp using AMPure XP beads (Beckman Coulter). A second 7-cycle PCR was performed using the same primers as for the first PCR. PCR product was purified by AMPure XP beads. Libraries were generated from two biological replicates, and sequenced using the Illumina HiSeq 1,500 platform.
Heilig A.K., Nakamura R., Shimada A., Hashimoto Y., Nakamura Y., Wittbrodt J., Takeda H, & Kawanishi T. (2022). Wnt11 acts on dermomyotome cells to guide epaxial myotome morphogenesis. eLife, 11, e71845.
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10

Fecal DNA Extraction and 16S rRNA Microbiome Sequencing

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DNA from feces for 16S rRNA gene-based metagenome analysis was isolated using the QIAamp DNA Stool Mini Kit (Qiagen N.V., Venlo, Netherlands) following the manufacturer’s protocol. Samples were incubated in the lysis buffer at 90 °C for 10 min for optimal bacterial lysis. For microbiome sequencing, DNA libraries were prepared using the Illumina Nextera Sample Preparation Kit with DNA primers corresponding to V3–V4 regions of the 16S rRNA. Illumina MiSeq was used for sequencing the libraries (Table 3).
Gromova L.V., Ermolenko E.I., Sepp A.L., Dmitrieva Y.V., Alekseeva A.S., Lavrenova N.S., Kotyleva M.P., Kramskaya T.A., Karaseva A.B., Suvorov A.N, & Gruzdkov A.A. (2021). Gut Digestive Function and Microbiome after Correction of Experimental Dysbiosis in Rats by Indigenous Bifidobacteria. Microorganisms, 9(3), 522.
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