Nextera xt dna sample preparation

Manufactured by Illumina

Sourced in United States

The Nextera XT DNA Sample Preparation kit is a library preparation system designed to generate sequencing-ready libraries from DNA samples. The kit utilizes a tagmentation process to simultaneously fragment and tag DNA, enabling rapid library construction. The core function of the Nextera XT kit is to prepare DNA samples for sequencing on Illumina's next-generation sequencing platforms.

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8 protocols using nextera xt dna sample preparation

TP53 Gene Sequencing Protocol

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Genomic DNA was isolated using the DNeasy Blood and Tissue kit (Qiagen) as recommended by the manufacturer. TP53 was amplified using three primer pairs (Supplementary Table 1), 50 ng of DNA and Taq Platinum (Invitrogen), and amplified products purified with Purelink genomic DNA purification kit (Invitrogen). Subsequently, DNA libraries were constructed with 1 ng of PCR product and Nextera XT DNA sample preparation (Illumina) following the manufacturer’s instructions and sequenced in a HiSeq 2500 platform. Reads were submitted to the above described quality control for cDNA reads. Good quality reads were mapped to the human genome with BWA aligner [18] .

Souza-Santos P.T., Soares Lima S.C., Nicolau-Neto P., Boroni M., Meireles Da Costa N., Brewer L., Menezes A.N., Furtado C., Moreira M.A., Seuanez H.N., de Almeida Simão T, & Ribeiro Pinto L.F. (2018). Mutations, Differential Gene Expression, and Chimeric Transcripts in Esophageal Squamous Cell Carcinoma Show High Heterogeneity. Translational Oncology, 11(6), 1283-1291.

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Analyzing Activated BMDC Transcriptome

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Day 9 differentiated and transduced BMDCs were activated with LPS for 0, 2, 4 and 6 hours for the RNA-seq experiments or for 8 hours before staining with Il-6, MIp1a, CD11c and CD14 antibody. Cells with gene specific sgRNAs were compared to those with non-targeting sgRNAs. For RNA purification we used Qiagen RNAeasy 96 Kit and constructed RNA libraries using the SMART-seq2 protocol (Picelli et al., 2013 (link)) in a 96 well plate format followed by Nextera XT DNA Sample Preparation (Illumina) and deep sequencing on a HiSeq 2500.

Parnas O., Jovanovic M., Eisenhaure T.M., Herbst R.H., Dixit A., Ye C.J., Przybylski D., Platt R.J., Tirosh I., Sanjana N.E., Shalem O., Satija R., Raychowdhury R., Mertins P., Carr S.A., Zhang F., Hacohen N, & Regev A. (2015). A genome-wide CRISPR screen in primary immune cells to dissect regulatory networks. Cell, 162(3), 675-686.

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Short-Read Illumina Sequencing Optimization

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The strains had previously been sequenced and assembled using Illumina short reads for our previous study [14 (link)], but to minimize biological disparities we opted to re-sequence with Illumina using the same DNA extraction prepared for long-read sequencing. Paired-end libraries were generated using the Nextera XT DNA sample preparation kit (Illumina), according to the manufacturer’s protocol. DNA was sequenced on a MiSeq sequencer (Illumina) with 150 bp reads for a theoretical read depth of 100×. Read quality metrics were evaluated using fastqc (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and fastp v0.19.6 [28 (link)]. Filterbytile from the BBMap package (http://sourceforge.net/projects/bbmap/) was used with default parameters for removing low-quality reads based on positional information on the sequencing flowcell and Trim Galore (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/), with settings --qual 20 and --length 126, provided additional adapter and quality trimming. fastq files were then randomly down-sampled to <100× crude read depth using an estimated genome size of 5.3 Mb, as higher read depths tend to reduce assembly quality [29 (link)].

Sydenham T.V., Overballe-Petersen S., Hasman H., Wexler H., Kemp M, & Justesen U.S. (2019). Complete hybrid genome assembly of clinical multidrug-resistant Bacteroides fragilis isolates enables comprehensive identification of antimicrobial-resistance genes and plasmids. Microbial Genomics, 5(11), e000312.

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Metagenomic DNA Extraction and Sequencing

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DNA extraction from the MFM sample was undertaken using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol.
The extracted DNA was quantified using the Qubit® 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and then subjected to next-generation sequencing. Libraries were generated according to the Nextera XT DNA Sample Preparation protocol (Illumina, San Diego, CA, USA). Normalized sample libraries were pooled and subjected to the cluster generation step using the cBOT cluster generation station, according to the Illumina TruSeq Paired-End Cluster Kit protocol. DNA sequencing was performed with the Illumina HiScanSQ sequencer, using the paired-end method and 93 bp of sequencing.

Tanca A., Palomba A., Pisanu S., Deligios M., Fraumene C., Manghina V., Pagnozzi D., Addis M.F, & Uzzau S. (2014). A straightforward and efficient analytical pipeline for metaproteome characterization. Microbiome, 2, 49.

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Genomic DNA Isolation and Sequencing

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For each isolate a single colony was transferred from BHI agar to 800 μL of BHI broth (Oxoid, Ottawa, ON, Canada) and incubated at 37°C for 3 h following which genomic DNA was isolated from 400 μL of broth culture using the Promega Maxwell^® 16 Cell DNA purification kit (Promega, Madison, WI, United States). Double-stranded genomic DNA was quantified using the Quant-iT^TM High Sensitivity Assay kit (Life Technologies Inc., Burlington, ON, Canada) according to the manufacturers’ recommendations. Sequencing libraries were constructed using the Nextera XT DNA sample preparation and the Nextera XT Index Kits (Illumina, Inc., San Diego, CA, United States) and paired-end sequencing was performed on the Illumina MiSeq platform, using 600-cycle MiSeq reagent kits (v3) with 5% PhiX control (Illumina Inc.).

Cooper A.L., Low A.J., Koziol A.G., Thomas M.C., Leclair D., Tamber S., Wong A., Blais B.W, & Carrillo C.D. (2020). Systematic Evaluation of Whole Genome Sequence-Based Predictions of Salmonella Serotype and Antimicrobial Resistance. Frontiers in Microbiology, 11, 549.

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Whole Transcriptome Amplification and Sequencing

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Whole transcriptome amplification of cell lysates was performed by SMART-Seq (Ramskold et al., 2012 ) using the Fluidigm C₁ Single-Cell Auto Prep System, followed by Nextera XT DNA Sample preparation (Illumina), as described in the Supplemental Experimental Procedures. We collected at least two independent biological replicates for each in vivo and in vitro condition, and two technical replicates for two in vivo conditions.

Gaublomme J.T., Yosef N., Lee Y., Gertner R.S., Yang L.V., Wu C., Pandolfi P.P., Mak T., Satija R., Shalek A.K., Kuchroo V.K., Park H, & Regev A. (2015). Single-cell Genomics Unveils Critical Regulators of Th17 cell Pathogenicity. Cell, 163(6), 1400-1412.

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Nuclei and Nucleoli Isolation and Sequencing

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Nuclei and nucleoli were isolated as previously described (Pontvianne et al. 2016a ) using a S3 cell sorter (Biorad®) at the Bioenvironment cytology platform (Perpignan University, Perpignan, France). Sorted nuclei or nucleoli were treated with RNase A and proteinase K prior to phenol/chloroform DNA purification, followed by two precipitation steps. DNA libraries were generated via the kit Nextera XT DNA sample preparation (Illumina®) according to manufacturer's instructions and were, then, subjected to high throughput paired-end 2X150nt sequencing on a Next-seq 550 apparatus (Illumina ®) at the Bioenvironment sequencing platform (Perpignan University, Perpignan, France). NADs identification was then performed as described in (Carpentier et al. 2018) (link). DNA-seq data are deposited at the ENA European Nucleotide Archive under the reference PRJEB36061.

Picart-Picolo A., Picart C., Picault N, & Pontvianne F. (2020). Nucleolus-associated chromatin domains are maintained under heat stress, despite nucleolar reorganization in Arabidopsis thaliana. Journal of plant research, 133(4).

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Nuclei and Nucleoli Isolation and Sequencing

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1 gram of leaves from 3-week-old FIB2:YFP plants are fixed for 20 min in 4% formaldehyde in Tris buffer (10 mM Tris-HCl at pH 7.5, 10 mM EDTA, 100 mM NaCl) and are then washed twice for 10 min in ice-cold Tris buffer. Washed leaves are minced with a razor blade in 1 mL of 45 mM MgCl2, 20 mM MOPS (pH 7.0), 30 mM sodium citrate, and 0.1% Triton X-100. The homogenate is filtered through 30μm mesh (PARTEC CellTrics) and subjected to FACS to sort nuclei or sonicated using a Bioruptor (three 5-min pulses, medium power; Diagenode) to liberate nucleoli that were then sorted by FANoS. Sorting of nuclei or nucleoli was triggered by the FIB2:YFP signal using a BD FACS Aria II. Sorted nuclei or nucleoli were treated with RNase A and proteinase K prior to purification and concentration using the kit ChIP DNA Clean & Concentrator (Zymo Research). DNA libraries were generated via the kit Nextera XT DNA sample preparation (Illumina®) according to manufacturer’s instruction, and were then subjected to high throughput paired-end 2X125nt sequencing on a Hiseq 2500 apparatus (Illumina ®). Around 40 million clusters were recovered from the sequencing for each sample.

Pontvianne F., Carpentier M.C., Durut N., Pavlištová V., Jaške K., Schořová Š., Parrinello H., Rohmer M., Pikaard C.S., Fojtová M., Fajkus J, & Saez-Vasquez J. (2016). Identification of nucleolus-associated chromatin domains reveals the role of the nucleolus in the 3D organisation of the A. thaliana genome. Cell reports, 16(6), 1574-1587.

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