One microgram of high-quality total RNA was used for RNA sequencing (RNA-seq) library construction using the TruSeq RNA Sample Preparation kit v2 (Illumina, San Diego, CA) with the following modification: one minute fragmentation time was applied to allow for longer RNA fragments. The obtained RNA-seq libraries were analyzed with the 2100 Bioanalyzer. The tissue sample described previously was then subjected to size selection and designated “size selected ”. Selection of sequences 375–900 nucleotides (nt) in length (275–800 nt sequences plus 50 nt sequencing adaptors on each end of the cDNA) was performed using the Pippin Prep system (Sage Science, Beverly, MA, USA). All libraries were then quantified with qPCR according to Illumina recommendations. The sequencing was performed at the Kansas State University Integrated Genomics Facility on the MiSeq personal sequencing system (Illumina) using the 600 cycles MiSeq reagent v3 kit (Illumina) according to Illumina instructions, resulting in 2 × 300 nt reads.
Miseq personal sequencing system
Manufactured by Illumina
Sourced in United States
The MiSeq Personal Sequencing System is a benchtop DNA sequencing instrument manufactured by Illumina. It is designed to perform targeted sequencing of specific genomic regions or small genomes. The system utilizes Illumina's proprietary sequencing-by-synthesis technology to generate high-quality sequencing data.
Automatically generated - may contain errors
Lab products found in correlation
Pippin prep system, by Sage Science (1 mentions)
Truseq rna sample preparation kit v2, by Illumina (1 mentions)
Miseq reagent kit v3, by Illumina (1 mentions)
Powerlyzer powersoil dna isolation kit, by Qiagen (1 mentions)
Miseq reagent kit v2, by Illumina (1 mentions)
Nextera xt dna sample preparation kit, by Illumina (1 mentions)
High sensitivity dna analysis kit, by Agilent Technologies (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Miseq reagent kit v2 500 cycle, by Illumina (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Nanodrop 8000, by Thermo Fisher Scientific (1 mentions)
Dneasy powersoil pro kit, by Qiagen (1 mentions)
Clc genomics workbench 20, by Qiagen (1 mentions)
5 protocols using miseq personal sequencing system
1
RNA Sequencing of Size-Selected Tissue
2
Soil Metagenomic Sequencing Protocol
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DNA was extracted from 250 mg (wet weight) of 57 soil samples (3 samples taken before the first fertilization + 3 samples x 6 experimental treatments x 3 applications of fertilizer) using the Power Lyzer Power Soil DNA Isolation Kit (Mo Bio Laboratories Inc., Carlsbad, CA, USA) according to the manufacturer’s instructions. The DNA extracts were stored at −20°C until use.
Soil DNA samples were used to prepare libraries using the MiSeq Reagent Kit v.2 (500 cycles; Illumina, San Diego, CA, USA) for shotgun metagenomic sequencing in a MiSeq Personal Sequencing System (Illumina, San Diego, CA, USA). In summary, we sequenced a subset of the original 57 samples and captured an average of 105.5 MB of genomic sequences per sample (S1 Table ).
Soil DNA samples were used to prepare libraries using the MiSeq Reagent Kit v.2 (500 cycles; Illumina, San Diego, CA, USA) for shotgun metagenomic sequencing in a MiSeq Personal Sequencing System (Illumina, San Diego, CA, USA). In summary, we sequenced a subset of the original 57 samples and captured an average of 105.5 MB of genomic sequences per sample (
3
Bacterial Genome Sequencing and Annotation
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Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. The preparation of genomic libraries was performed with 1 ng of genomic DNA using the Nextera XT DNA Sample Preparation Kit (Illumina Inc., CA) according to the manufacturer's instructions. After PCR amplification and cleanup, the fragment size distribution of the tagmented DNA was analysed using an Agilent 2100 Bioanalyzer and the High Sensitivity DNA Analysis Kit (Agilent Technologies, Santa Clara, CA). The libraries were sequenced using a MiSeq Personal Sequencing System and the MiSeq Reagent Kit v2 (500 cycles) (Illumina Inc.). Quality trimming and de novo assembly of the raw paired-end reads were performed using the CLC Genomics Workbench (v 6.0) software package (CLC bio, Aarhus, Denmark) with default settings, except for contig length (minimum contig length = 2,000 bp). Seventeen genomes that were sequenced de novo in this study were automatically annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) 2.0 and were manually checked as part of the process of genome submission to GenBank. Our sequencing efforts resulted in multiple contigs (Table 1 ). The datasets for the genome annotations for the other 32 strains published previously were retrieved from the FTP server of NCBI [20 ].
4
Soil Microbiome DNA Sequencing Protocol
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Soil samples for DNA analysis were flash-frozen and kept at −20 °C until analysis. DNA from frozen soil samples was isolated using the DNeasy PowerSoil Pro Kit (Qiagen, Germany), according to the manufacturer’s instructions. The quality and concentration of DNA were assessed spectrophotometrically by a NanoDrop™ 8000 instrument (ThermoFischer Scientific, Waltham, MA, USA). Two nanograms of DNA were used as an input for the library preparation. The amplicon libraries of the hypervariable V4 region of the 16S rRNA gene were prepared using a two-stage PCR strategy with the primers 515F [32 (link)] and Pro-mod-805R [33 (link)]. PCR of every DNA sample was carried out in duplicate; the detailed amplification protocol is described previously [34 (link)]. The libraries were checked with agarose gel and pooled equimolarly. The final pool was purified with the Cleanup Mini PCR Purification Kit (Evrogen, Russia) according to the manufacturer’s instructions. Sequencing was performed with the MiSeq™ Personal Sequencing System (Illumina, San Diego, CA, USA) using the 156-bp paired-end reads.
5
Sequencing and Bioinformatic Analysis
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Sequencing was performed using the MiSeq™ Personal Sequencing System (Illumina, San Diego, CA, USA) with 2 × 156 bp paired end. The ‘Trim reads’ tool in CLC Genomics Workbench 20.0 (Qiagen, Hilden, Germany) was used to efficiently filter out and remove nucleotides corresponding to primers 515F and Pro-mod-805R. Demultiplexing was performed using the deML package [45 (link)], using zero available mismatches option. Then, read pairs were processed with DADA2 pipeline [46 (link)], according to published.
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