Sequencing libraries were prepared with the 16S Metagenomic Sequencing Library Preparation guide (Illumina) using the Nextera XT index kit. DNA isolated for the CFU assay was used for PCR amplification with Q5 polymerase (New England Biolabs). Thermocycling conditions were 30 s at 98 °C for denaturation, followed by 24 cycles of (7 s at 98 °C, 12 s at 69 °C, 15 s at 72 °C) and a final extension for 2 min at 72 °C. Amplifications were performed twice to generate technical replicates. Index PCR was performed with Q5 polymerase and the Nextera XT index kit before Illumina Next Generation Sequencing (MiSeq Nano V2, 250 bp paired end reads).
Miseq nano v2
Manufactured by Illumina
Sourced in United States
The MiSeq Nano V2 is a benchtop DNA sequencing system designed for small-scale sequencing projects. It utilizes Illumina's proprietary sequencing-by-synthesis technology to generate high-quality DNA sequence data. The system is capable of sequencing a wide range of sample types, including genomic DNA, amplicons, and microbiome samples. The MiSeq Nano V2 is intended to provide researchers with a compact and efficient solution for their sequencing needs.
Automatically generated - may contain errors
Lab products found in correlation
Nextera xt index kit, by Illumina (1 mentions)
Q5 polymerase, by New England Biolabs (1 mentions)
Q5 polymerase, by Illumina (1 mentions)
16s metagenomic sequencing library preparation guide, by Illumina (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Nebnext ultra 2 directional rna library prep kit, by New England Biolabs (1 mentions)
Rnase free dnase 1, by Roche (1 mentions)
Novaseq s1 flow cell, by Illumina (1 mentions)
Nebnext poly a mrna magnetic isolation module, by New England Biolabs (1 mentions)
Novaseq 6000 platform, by Illumina (1 mentions)
Minielute pcr purification kit, by Qiagen (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
Pfuultra 2 hotstart pcr master mix, by Agilent Technologies (1 mentions)
Maxima h minus first strand dna synthesis kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using miseq nano v2
1
16S Metagenomic Sequencing Library Preparation
2
Genome Sequencing and Annotation of M. blatticola
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The M. blatticola PA genome was sequenced from DNA ordered at the DSMZ German culture collection (DSM 13328), using Illumina MiSeq Nano V2 (2 ×250 PE). Reads were assembled using Spades 3.11 [24 (link)]. A total of 12 contigs >1 kb were obtained, with an average coverage of 157×. Almost all reads were assembled, as 99.97% of them aligned on these contigs. Genes were predicted using prodigal [25 (link)]. The genome and protein sequences are available in GenBank, PRJNA731512, and in Supplementary Dataset 1 and 2 . Gene functions were annotated with Kyoto Encyclopedia of Genes and Genomes (KEGG) [26 (link)] and Eggnog [27 (link)]. Genes associated with the methanogenesis pathways were specifically targeted with HMM searches using PFAM [28 ], TIGRFAMs [29 (link)], and custom HMM profiles. Presence of transmembrane domains was determined using TMHMM 2.0 [30 (link)]. In the course of our analysis, this strain was also sequenced by JGI (2756170388) and released in GenBank (GCA_004363215.1). Unsurprisingly, the sequences and statistics of the two genomes are practically identical (size difference of <1 kb, average nucleotide identity (ANI) of 99.99%, 13 contigs). The analyses presented here were conducted on the genome we sequenced.
3
Transcriptome Profiling of Primary Macrophages
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Total RNA was extracted from 2 × 106 PMφs using an RNeasy mini kit (Qiagen, no. 74104). To eliminate genomic DNA, 20 U of Roche RNase-free DNase I (Roche, no. 4716728001) was added in the eluant (100 µl), followed by 20 min of incubation at 37°C. Samples were then passed through RNeasy columns. Total RNA was quantified by a NanoDrop ND-1000 spectrophotometer, and 750 ng was used to enrich mRNA using an NEBNext poly(A) mRNA magnetic isolation module (New England Biolabs, no. E7490). Library construction followed immediately using an NEBNext Ultra II directional RNA library prep kit (New England Biolabs, no. E7760) and NEBNext multiple oligonucleotides for Illumina (New England Biolabs, nos. E7335, E7500, E7710, E7730). Libraries were PCR amplified for 10 cycles, quantified by MiSeq Nano v2, and sequenced on an Illumina NovaSeq S1 flow cell with a 150-bp paired-end run to obtain ∼30 million reads per sample. The complete RNA sequencing (RNA-seq) dataset has been deposited to the GEO repository (GSE239696; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE239696 ).
4
Soil DNA Extraction and Microbial Sequencing
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DNA extractions for 2019 samples were performed using a modified protocol to extract DNA and RNA from soil using phenol–chloroform methods (see Supplemental Materials S1.1) in Urbana, IL. Details for DNA extractions methods for 2014 samples are given in detail in Schmitt et al. (2019). The quality and quantity of extracted DNA were determined by agarose gel electrophoresis and fluorometry (Qubit 4.0). Primer sets for bacterial- and archaeal 16S rRNA genes, N- and S-cycle genes of interest (narG, nxrB, nirK, nirS, nosZ, nifH, amoA, amoB, nrfA, dsrB, aprA, soxB) were used to generate sequence libraries (Table S1). Specific gene-targeted amplicons for downstream sequencing were generated for all samples using a Fluidigm microarray system through services available in the Functional Genomics Unit of the University of Illinois Urbana-Champaign Carver Biotechnology Center (Fluidigm Array details in Supplemental Materials S1.2). Paired-end sequencing (2 × 250 base pairs) was performed on one lane of a MiSeq Nano (v2) (San Diego, CA, USA) for 2014 samples and one lane of an Illumina NovaSeq 6000 platform (San Diego, CA, USA) for 2019 samples. Raw sequence data were deposited in MG-RAST (2014 samples, project number MGP82583) and NCBI (2019 samples, BioProject ID PRJNA76940).
5
SHAPE-MaP Analysis of PAN RNA
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Each reaction included 1 × 107 BCBL-1 cells washed with 1× PBS, collected by centrifugation at 300g for 5 min, and resuspended in 900 µL of RPMI-1640 medium. The SHAPE (+) reaction included the addition of 100 µL of 100 mM 1-methyl-7-nitroisatoic anhydride (1M7, DC Chemicals DC8649) resuspended in 100% DMSO to a final concentration of 10 mM to 900 µL of cells, followed by the incubation for 5 min at 37°C. For the SHAPE (−) reaction, 100 µL of 100% DMSO was added to 900 µL of cells and incubated for 5 min at 37°C. The denatured control (dc) reaction included 1 µg/µL of total RNA extracted from BCBL-1 cells, which was combined with 5 µL 100% formamide and 1 μL 10× denaturing buffer (500 mM HEPES pH 8.0, 40 mM EDTA) in a total volume of 9 μL and incubated for 1 min at 95°C. An amount of 1 µL of 100 mM 1M7 was subsequently added to 9 µL of the sample and incubated for 1 min at 95°C. SHAPE-MaP libraries were constructed by dividing the PAN RNA sequence into three overlapping zones, including nt 15–343 for zone 1, nt 311–707 for zone 2, and nt 694–1060 for zone 2 (Supplemental Table 1 ). Libraries were sequenced using MiSeq Nano V2 (Illumina) paired-end sequencing 2 × 250 bp with on average 100,000 reads per sample. SHAPE-MaP data were analyzed using the ShapeMapper v1.2 pipeline (21).
6
Dengue Virus Type 2 Genome Sequencing
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Dengue virus type 2, New Guinea C (Culture collection) RNA was extracted from aliquoted culture supernatants using QIAamp Viral RNA mini kit (Qiagen). cDNA was synthesized using Maxima H Minus First Strand DNA synthesis kit (Thermo Scientific) with random hexamer primers, according to manufacturer’s instructions. The cDNA was amplified in seven 1700–2500 bp overlapping amplicons using PfuUltra II Hotstart PCR Master Mix (Agilent Technologies). PCR products were separated in a 1% agarose TAE gel. After confirming the bands of interest were present without non-specific products, the PCR product was purified using the MIniElute PCR purification kit (Qiagen). Sequencing libraries were prepared using an in-house (INCPM) DNA-Seq protocol, and sequenced 2 × 150 on an Illumina MiSeq nano v2.
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