Metagenomic DNA extracts as well as sorted single cell MDA products were quantified using the Qubit dsDNA HS Assay Kit (ThermoFisher Scientific, OR, United States). Illumina Sequencing libraries were then prepared using the NEBNext® UltraTM DNA Library Prep Kit and NEBNext® UltraTM II FS DNA Library Prep Kit (New England BioLabs, Frankfurt, Germany), respectively, using unique dual index adapters and following the manufacturer’s instruction. Resulting library fragment lengths were assessed using the Agilent 2100 Bioanalyzer with a High Sensitivity DNA Kit (Agilent Technologies, Germany). The libraries were then pooled and sequenced on an Illumina NovaSeq system using a paired-end approach with 150 cycles per read. Sequencing depths of approximately 6.8 million read pairs or 2 Gb were produced per SAG, on average.
Nebnext ultra 2 fs dna library prep kit
Manufactured by New England Biolabs
Sourced in United States, United Kingdom
The NEBNext Ultra II FS DNA Library Prep Kit is a library preparation kit designed for generating DNA libraries for next-generation sequencing. The kit utilizes a streamlined workflow to convert DNA samples into Illumina-compatible libraries.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (6 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (5 mentions)
Novaseq 6000, by Illumina (5 mentions)
Nextseq 550 system, by Illumina (5 mentions)
Microbexpress kit, by Thermo Fisher Scientific (4 mentions)
2100 bioanalyzer, by Agilent Technologies (4 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
High sensitivity dna kit, by Agilent Technologies (3 mentions)
Nebnext rrna depletion kit for human mouse rat, by New England Biolabs (3 mentions)
Nebnext ultra 2 direction rna sequencing prep kit, by New England Biolabs (3 mentions)
Hiseq x ten platform, by Illumina (3 mentions)
Novaseq 6000 platform, by Illumina (3 mentions)
Hiseq 2500, by Illumina (3 mentions)
Nextseq 500, by Illumina (3 mentions)
Nebnext ultra dna library prep kit, by New England Biolabs (2 mentions)
Agilent high sensitivity dna kit, by Agilent Technologies (2 mentions)
Qubit dsdna hs kit, by Thermo Fisher Scientific (2 mentions)
Agilent high sensitivity d5000 screentape system, by Agilent Technologies (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Nextseq 500 550 high output kits v2, by Illumina (2 mentions)
66 protocols using nebnext ultra 2 fs dna library prep kit
1
High-throughput single-cell genome sequencing
2
Quantification, Library Prep, and Sequencing of Genomic DNA from WWTP Samples
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Genomic DNA extracted from the WWTP samples and MDA products was quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, OR, United States). Libraries were prepared using the NEBNext® UltraTM DNA Library Prep Kit and NEBNext® UltraTM II FS DNA Library Prep Kit (New England BioLabs, Frankfurt, Germany), respectively, following the manufacturer’s instruction. Five hundred nanogram of DNA was used as starting material. The quality of the DNA libraries was verified using the Agilent High Sensitivity DNA Kit on the Agilent 2100 Bioanalyzer instrument (Agilent Technologies, Germany). The libraries were then pooled and sequenced on Illumina systems using the paired-end approach and the highest available read length for each platform (150 bp for NovoSeq and NextSeq, 300 bp for MiSeq). Illumina platforms used to sequence SAGs are listed in Supplementary Table 6 .
3
Enriching Mansonella DNA for Sequencing
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The NEBNext Microbiome DNA enrichment kit was used as directed (New England Biolabs Inc., USA) to enrich Mansonella DNA and reduce human DNA contamination prior to library construction. The Illumina libraries were constructed for M. perstans and M. ozzardi using the NEBNext Ultra II FS DNA Library Prep Kit (New England Biolabs Inc., USA) as described by the manufacturer. For M. perstans, 2 ng of DNA was fragmented with the FS enzyme mix for 10, 20 or 30 minutes, and for M. ozzardi, 50 ng of DNA was fragmented for 5, 10 or 20 minutes. A library was prepared from each time point then PCR amplified with a different index primer to enable multiplexing. The quality and concentration of each library was determined using a 2100 Bioanalyzer with a high sensitivity DNA chip (Agilent Technologies, USA). Libraries were diluted to 4 nM with 10 mM Tris, 0.1 mM EDTA pH 8, and equal volumes of the 3 libraries from each species were mixed to create a pooled sample. Phi X DNA was added to balance base pair composition in these A:T rich filarial libraries prior to sequencing on a NextSeq500 platform (paired end, 150 bps).
4
Fosmid Library Preparation and Sequencing
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Library preparation of fosmid-DNA was performed using the NEBnext Ultra II FS DNA Library Prep Kit (New England BioLabs, Ipswich, MA, USA) following the manufacturer’s protocol for <100 ng DNA input (30–46 ng input amount). The quality was controlled using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and the libraries were subsequently sequenced on an Illumina MiSeq (Illumina, San Diego, CA, USA) with a 600v3 sequencing kit (2 × 300 bp paired-end reads). The sequence data were trimmed and quality controlled using fastp [30 (link)], and de novo assembled in the CLC Genomics Workbench (version 10; QIAGEN, Hilden, Germany). Sequence annotations were transferred from the reference vector constructs using Geneious Prime (Biomatters, Auckland, New Zealand). The CeHV-2 sequence coverage was above 99% for all sequenced fosmids and sequences were identical to the reference genome (Genbank NC_006560).
5
Metagenomics and Metatranscriptomics Library Prep
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Libraries for metagenomics and metatranscriptomics were prepared as described in our recent work (Cabral et al., 2020 (link)). We prepared metagenomic libraries from DNA (100 ng) using the NEBNext® Ultra II FS DNA Library Prep Kit (New England BioLabs, Ipswich, MA, USA) and the > 100 ng input protocol as per the manufacturer’s instructions, which generated a pool of fragments whose average size was between 250 and 500 bp. Meanwhile, we prepared metatranscriptomic libraries from total RNA (≤1 ug) using a combination of the MICROBExpress kit (Invitrogen, Carlsbad, CA, USA), NEBNext® rRNA Depletion Kit for Human/Mouse/Rat (New England BioLabs, Ipswich, MA, USA), and the NEBNext® Ultra II Direction RNA Sequencing Prep Kit as per the manufacturers’ instructions. This generated a pool of fragments with an average size between 200 and 450 bp. Both metagenomic and metatranscriptomic libraries were pair-end sequenced (2×150 bp) on the Illumina HiSeq X Ten platform, yielding an average of 1,464,061 ± 728,330 reads per metagenomic sample and 35,884,874 ± 27,059,402 reads per metatranscriptomic sample.
6
Genome-wide CRISPR Off-target Analysis
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Genome-wide, unbiased off-target analysis was performed following the iGUIDE pipeline based on Guide-seq invented previously (62 (link),63 (link)). HEK293T cells were transfected in 20 uL Lonza SF Cell Line Nucleofector Solution on a Lonza Nucleofector 4D with program DS-150 according to the manufacturer's instructions. 300 ng of gRNA–Cas9 plasmids (or 150 ng of each gRNA–Cas9n plasmid for the double nickase), 150 ng of the effector plasmids, and 5 pmol of double stranded oligonucleotides (dsODN) were transfected. Cells were harvested after 72 h for genomic DNA using Agencourt DNAdvance reagent kit. 400 ng of purified gDNA which was then fragmented to an average of 500 bp and ligated with adaptors using NEBNext Ultra II FS DNA Library Prep kit following manufacturer's instructions. Two rounds of nested anchored PCR from the oligo tag to the ligated adaptor sequence were performed to amplify targeted DNA, and the amplified library was purified, size-selected and sequenced using Illumina Miseq V2 PE300 or V3 PE600. Sequencing data was analyzed using the published iGUIDE pipeline, with the addition of a downsampling step which ensures an unbiased comparison across samples.
7
Targeted Sequencing of Key Oncogenes
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DNA fragmentation and library preparation were performed using the NEBNext Ultra II FS DNA library prep kit (New England Biolabs, USA) following the manufacturer’s instructions. DNA library concentrations were quantified with a QuantiFlour dsDNA system (Promega, USA). Equal amounts of libraries (150 ng per sample) were pooled together and hybridized with xGen Lockdown probes for six targeted genes EGFR, KRAS, NRAS, BRAF, ALK and ROS1 (IDT DNA, USA). For ALK and ROS1, customized probes (Table S3 ) for intron regions were designed and mixed with probes for exon regions at equal concentration. Sequencing was run using NextSeq. 500/550 High output kits v2 (150 cycles) on Illumina NextSeq. 550 system (Illumina, USA) with minimum target coverage of 100×. In cases where the mean coverage in the targeted regions is lower than 100×, extra sequencing was performed to increase the mean coverage to the expected range. The mean coverage in the target regions for all samples is approximately 129×.
8
SARS-CoV-2 Genome Sequencing Protocol
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SARS-CoV-2 RNA was extracted and purified as described in the “Determination of SARS-CoV-2 RNA titers” section. RT-PCR was used to generate five overlapping amplicons, and the NEBNext Ultra II FS DNA Library Prep Kit (New England BioLabs, Ipswich, MA, USA) was used for library preparations. NGS analysis was done as described (14 (link), 17 (link), 27 (link), 30 (link)).
9
DNA Library Prep from Micro-dissected Tissues
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DNA library preparation of micro-dissected tissue samples was undertaken using a bespoke low-input enzymatic-fragmentation-based library preparation method2 (link)–4 (link),37 (link). This method was employed as it allows for high quality DNA library preparation from a very low starting quantity of material (from 100-500 cells). In brief, gDNA was purified from cell lysates using bead purification. Enzymatic fragmentation, end-repair and dA-tailing was performed using NEBNext Ultra II FS DNA Library Prep Kit (New England BioLabs). Indexing and PCR amplification was subsequently performed (12 cycles). DNA library concentration was assessed after library preparation and used to guide choice of samples to take forward to DNA sequencing. The minimum library concentration was 5 ng/µL and libraries with >15 ng/µL were preferentially chosen. 150 bp paired-end Illumina reads were prepared with Unique Dual Index barcodes (Illumina). DNA sequencing was undertaken on a NovaSeq 6000 platform using an XP kit (Illumina). Samples were multiplexed in pools of 6-24 samples. Pools were sequenced to achieve a coverage of ~30x per sample.
10
Isolation and Sequencing of Phage Virions from Feces
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To study phage virions, we isolated the faecal VLP fraction and sequenced dsDNA phages as previously described19 (link). Briefly, the VLPs were extracted from 500 mg of faeces using high-speed centrifugation followed by filtration through a 0.45 µm membrane. Any free-DNA debris was digested prior to lysing the VLPs, whereafter the DNA was purified using a two-step phenol/chloroform extraction protocol. Finally, the DNA was purified using the DNeasy Blood&Tissue kit (Qiagen Cat#69506) according to the manufacturer’s protocol. Library preparation was done with the NEBNext Ultra II FS DNA library prep kit (New England Biolabs Cat#E7805L) and the NEBNext Multiplex Oligos for Illumina dual indexes (New England Biolabs Cat#E7600S) according to manufacturer’s instructions. Quality and concentration of the VLP libraries were assessed with the Qubit dsDNA HS kit (ThermoFisher Cat#Q32854) and with the Agilent High Sensitivity D5000 ScreenTape system (Agilent Technologies). Libraries were sequenced using 2×150 bp paired-end chemistry on an Illumina NovaSeq 6000 platform with the S4 Reagent Kit v1.5, 300 cycles (Illumina Cat#20028312) at the Core Facility Genomics of the Amsterdam UMC.
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