The total microbial genomic DNA was extracted from 1 to 5 g of AMD sediment samples using a FastDNA Spin Kit (MP Biomedicals, USA) according to the manufacturer’s instructions. For each sample, multiple extractions were performed, using 500 mg of sediment as the input for one extraction. Then, the DNA from the multiple extractions were combined. The extracted DNA yield and purity were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). The DNA library of each sample was constructed using a NEBNext Ultra II DNA Prep Kit (New England Biolabs, USA) following the manufacturer’s instructions. A total of 86 DNA libraries were successfully constructed, and they were sequenced on an Illumina HiSeq 2500 platform in the PE150 mode (Illumina, USA).
Nebnext ultra 2 dna prep kit
Manufactured by New England Biolabs
Sourced in United States
The NEBNext Ultra II DNA Prep Kit is a DNA library preparation kit designed for efficient and high-quality library construction from a variety of input DNA sources. The kit includes reagents and enzymes necessary for end-repair, adaptor ligation, and PCR amplification of DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Fastdna spin kit, by MP Biomedicals (3 mentions)
Agilent bioanalyzer 2100 dna 1000 kit, by Agilent Technologies (3 mentions)
Hiseq 2500 platform, by Illumina (2 mentions)
Qiaquick gel extraction kit, by Qiagen (2 mentions)
Miseq platform, by Illumina (1 mentions)
Miseq reagent kit v3, by Illumina (1 mentions)
150 bp paired end reads, by Illumina (1 mentions)
Hiseq 3000, by Illumina (1 mentions)
E220 focused ultrasonicator, by Covaris (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
Nebnext ultra 2 directional rna library prep kit, by New England Biolabs (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
7 protocols using nebnext ultra 2 dna prep kit
1
Microbial DNA Extraction and Sequencing
2
Metagenomic DNA Extraction and Sequencing
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Total DNA was extracted from 10 g of each sediment which was pretreated with 30 mL solution containing 0.1 mol/L ethylene diamine tetraacetic acid (EDTA), 0.1 mol/L Tris (pH 8.0), 1.5 mol/L NaCl, and 0.1 mol/L NaH2PO4 and Na2HPO4 prior to the employment of the FastDNA Spin Kit (MP Biomedicals, Irvine, CA)24 (link),64 (link). Extracted DNA was purified using the QIAquick Gel Extraction Kit (Qiagen, Chatsworth, CA). Finally, a total of 90 samples (with the other samples being discarded due to their low DNA yield/quality) were used for library preparation with NEBNext Ultra II DNA Prep Kit (New England Biolabs, MA) and sequenced from both ends with MiSeq Reagent Kit v3 on an Illumina MiSeq platform (150 bp, paired end reads). This generated totally ~7 Tb metagenomic raw reads data.
3
Microbial Community DNA Extraction and Sequencing
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DNA was extracted using FastDNA Spin kit (MP Biomedicals, Irvine, CA, United States) and purified with QIA quick Gel Extraction Kit (Qiagen, Chatsworth, CA, United States) according to manufacturer’s instructions. The quality and quantity of total community DNA were estimated using agarose gel electrophoresis and Qubit (Thermo Fisher Scientific, Australia). Finally, library preparation of DNA was conducted with NEBNext Ultra II DNA Prep Kit (New England Biolabs, Ipswich, MA, United States) and sequenced using an Illumina Hiseq2500 platform using a 150bp paired-end approach.
4
Targeted Sequencing of Populus and Salix
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Libraries for two individuals from each P. balsamifera L., P. tremula, P. mexicana Wesm., S. nigra Marshall, S. exigua Nutt., and S. phlebophylla Andersson (Appendix S2 ) were prepared using the NEBNext Ultra II DNA Prep Kit (New England Biolabs, Ipswich, Massachusetts, USA) following the manufacturer’s protocol, and quantified using an Agilent Bioanalyzer 2100 DNA 1000 kit (Agilent Technologies, Santa Clara, California, USA). Libraries were pooled at equimolar concentrations into two pools of six prior to probe hybridization following the Arbor Biosciences myBaits protocol version 3.0.1 and Hale et al. (2020 ). The hybridized samples were subsequently pooled at equimolar ratios and sequenced at the Texas Tech Center for Biotechnology and Genomics using a MiSeq with the v2 Micro kit and 150‐bp paired‐end reads (Illumina, San Diego, California, USA).
5
Targeted Sequencing of Populus and Salix
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Libraries for two individuals from each Populus balsamifera L., P. tremula L., P. mexicana Wesmael., Salix nigra Marshall, S. exigua Nutt., and S. phlebophylla Andersson (Table S3) were prepared using the NEBNext Ultra II DNA Prep Kit following the published protocol for this kit (New England Biolabs, Ipswitch, MA, USA), and quantified using an Agilent Bioanalyzer 2100 DNA 1000 kit (Agilent Technologies, Santa Clara, CA, USA). Libraries were pooled at equimolar concentrations into two pools of six prior to probe hybridization following the Arbor Biosciences myBaits protocol v 3.0.1 and Hale et al. (2020) . The hybridized samples were subsequently pooled at equimolar ratios and sequenced at the Texas Tech Center for Biotechnology and Genomics using a MiSeq with the Micro chemistry and 150 bp paired-end reads (Illumina, Inc., San Diego, CA, USA).
6
Sex Identification in Black Willow
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The sex of 24 males and 24 females of S. nigra was identified from flowering catkins in a wild population near Dickens Springs, TX, USA in April 2017. Leaf tissue was collected and dried on silica beads. DNA was extracted from leaf tissue using the Qiagen DNeasy Plant Minikit (Qiagen, Hilden, Germany) and fragmented using sonication with the Covaris E220 Focused Ultrasonicator (Covaris, Inc., Woburn, MA, USA). Libraries were prepared using the NEBNext Ultra II DNA Prep Kit (New England Biolabs, Ipswitch, MA, USA), and quantified using an Agilent Bioanalyzer 2100 DNA 1000 kit (Agilent Technologies, Santa Clara, CA, USA). Libraries were pooled at equimolar concentrations into sixteen pools of six prior to probe hybridization to targeted capture probes following the Arbor Biosciences myProbes protocol v 3.0.1. The hybridized samples were subsequently pooled at equimolar ratios and sequenced at the Oklahoma Medical Research Foundation (Oklahoma, OK, USA) using a HiSeq 3000 (Illumina, Inc., San Diego, CA, USA).
7
Comprehensive Genomic Profiling for Cancer
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A total of 571 genes related to DNA mutations (single nucleotide variant [SNV], indel, fusion, copy number variant [CNV], microsatellite instability, and tumor mutation burden) and 2660 genes for RNA expression and fusion detection (Supplementary Table 2) were analyzed using the AmoyDx Master Panel (Amoy Diagnostics, Xiamen, China). DNA and RNA library construction with NEBNext Ultra II DNA Prep Kit and NEBNext Ultra II Directional RNA Library Prep Kit (NEB, MA, USA), respectively was followed by targeted DNA and RNA sequencing based on the Illumina NovaSeq 6000 instrument (Illumina, San Diego, CA, USA), as previously described . For NTRK DNA rearrangement evaluation, the positive cut-off value was double-strand base calibration (DSBC) reads ≥3 for known hotspot fusion and DSBC reads ≥4 and variant allele frequency ≥0.4% for novel NTRK fusion, respectively. For NTRK RNA fusion assessment, the positive cut-off value was junction reads ≥2 for known hotspot fusion and junction reads ≥10 for novel NTRK fusion.
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