Genomic DNA (gDNA) extractions were performed using the Wizard Genomic DNA Purification Kit (Promega) following pre-treatment of S. aureus cells with 10 μg/ml lysostaphin (Ambi Products LLC) at 37°C for 30 min. The genome sequencing for NE202 (pgl) was performed by MicrobesNG using an Illumina HiSeq platform and a 250-bp paired end read kit. DNA libraries for pgl::Kmr and pglR1 were prepared using an Illumina Nextera XT DNA Library Prep kit, validating size distribution by gel electrophoresis, and bead-normalizing the libraries. An Illumina MiSeq v2 600 cycle kit was used for genome sequencing, generating 300-bp paired end reads. PhiX was used as a sequencer loading control. The CLC Genomics Workbench software (Qiagen Version 20) was used for genome sequencing analysis of the different strains, as described previously [91 (link)]. As a reference genome, a contig was produced for wild-type JE2 by mapping Illumina reads onto the closely related USA300 FPR3757 genome sequence (RefSeq accession number NC_007793.1). The Illumina short read sequences from NTML mutants [57 (link)] of interest were then mapped onto the assembled JE2 sequence, and the presence of the transposon insertion confirmed. Single Nucleotide Polymorphisms (SNPs), deletions or insertions were identified where present.
Miseq v2 600 cycle kit
Manufactured by Illumina
Sourced in United States
The MiSeq v2 600 cycle kit is a sequencing reagent kit designed for the Illumina MiSeq platform. The kit enables the generation of up to 600 sequencing cycles, providing extended read lengths for various applications that require high-quality, long-read data.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq platform, by Illumina (2 mentions)
Nextera xt dna library prep kit, by Illumina (2 mentions)
Clc genomics workbench software, by Qiagen (2 mentions)
Wizard genomic dna purification kit, by Promega (2 mentions)
Rneasy plant mini kit, by Qiagen (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
7 protocols using miseq v2 600 cycle kit
1
Genomic DNA Extraction and Sequencing of S. aureus
2
Illumina MiSeq Sequencing Library Preparation
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The purified amplicons were subjected to an Index PCR step that allows for the labelling of the samples by attaching dual indices and adapters. For this, 5 µL of sample was mixed with 25 µL of 2X KAPA HiFi HotStart ReadyMix, 5µL of Nextera XT Index 1 Primers (i5), 5 µL Nextera XT Index 2 Primers (i7), and 10 µL of PCR-grade water. PCR conditions were as follows: initial denaturation at 95 °C for 3 min; 8 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; final extension at 72 °C for 5 min. This was followed by a new cleaning step of the final library with ethanol 80%. The final step included libraries quantification using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). All libraries were pooled in equal molar ratios and denaturated with 0.2 N NaOH for sequencing. Samples were sequenced using an Illumina MiSeq v2 600 cycle kit (Illumina, San Diego, CA, USA).
3
Phage Genome Sequencing Protocol
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Phage samples from individual time points during the ABE8e evolution campaign were used for high-throughput sequencing. 1 uL of each sample was used for PCR with primers KZ1532 (5’-ATAAACTGATACAATTAAAGGCTCC-3’) and KZ1533 (5’-GGTGTTTCGCTACCGGAAGAACCAC-3’) to yield PCR products of 602 base pairs in length. PCR activation was done at 95 °C for 10 min to ensure phage lysis, followed by 30 cycles of 30 second extensions. After each sample was confirmed by agarose gel, a second round of PCR for 10 additional cycles was performed to barcode each sample individually. All samples were then pooled at comparable amounts and sequenced using an Illumina MiSeq v2 600-cycle kit.
4
Phage Genome Sequencing Protocol
5
Genomic DNA Extraction and Sequencing
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Genomic DNA (gDNA) extractions were performed using the Wizard® Genomic DNA Purification Kit (Promega) following pre-treatment of S. aureus cells with 10 μg/ml lysostaphin (Ambi Products LLC) at 37°C for 30 min. The genome sequencing for NE202 (pgl) was performed by MicrobesNG using an Illumina HiSeq platform and a 250-bp paired end read kit. DNA libraries for pgl::Kmr and pglR1 were prepared using an Illumina Nextera XT DNA Library Prep kit, validating size distribution by gel electrophoresis, and bead-normalizing the libraries. An Illumina MiSeq v2 600 cycle kit was used for genome sequencing, generating 300-bp paired end reads. PhiX was used as a sequencer loading control. The CLC Genomics Workbench software (Qiagen Version 20) was used for genome sequencing analysis of the different strains, as described previously (89 (link)). As a reference genome, a contig was produced for wild-type JE2 by mapping Illumina reads onto the closely related USA300 FPR3757 genome sequence (RefSeq accession number NC_007793.1). The Illumina short read sequences from NTML mutants (57 (link)) of interest were then mapped onto the assembled JE2 sequence, and the presence of the transposon insertion confirmed. Single Nucleotide Polymorphisms (SNPs), deletions or insertions were identified where present.
6
High-throughput Sequencing of Plant Virus Isolates
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Sample references 76/16 and 77/48: A subset of the original samples collected from Cambridge, England, had been stored dried over Calcium chloride and were deposited in the collection of the late Dr Alan Brunt. This collection was subsequently deposited with The University of Warwick. Two isolates labelled PlVX 76/16 and 77/48 were subsampled, and the subsamples were sent to Fera Science Ltd, York, UK, for high throughput sequencing by a ribosomal RNA-depleted total RNA approach. (see Supplementary information S.1). RNA was extracted from the two sub-samples using an RNeasy plant mini kit (Qiagen, UK). Indexed TruSeq complete plant libraries, including RNA depletion step, were produced from the RNA and sequenced using a MiSeq V2 600 cycle kit (Illumina). The resulting data was then analyzed as described in Fox et al. (2019) . Phylogenetic trees were produced using the Maximum likelihood algorithm and 500 bootstraps in MEGA 7 (Kumar et al., 2016) . Pairwise identities were calculated using the same software.
7
High-throughput Sequencing of Plant Virus Isolates
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Sample references 76/16 and 77/48: A subset of the original samples collected from Cambridge, England, had been stored dried over Calcium chloride and were deposited in the collection of the late Dr Alan Brunt. This collection was subsequently deposited with The University of Warwick. Two isolates labelled PlVX 76/16 and 77/48 were subsampled, and the subsamples were sent to Fera Science Ltd, York, UK, for high throughput sequencing by a ribosomal RNA-depleted total RNA approach. (see Supplementary information S.1). RNA was extracted from the two sub-samples using an RNeasy plant mini kit (Qiagen, UK). Indexed TruSeq complete plant libraries, including RNA depletion step, were produced from the RNA and sequenced using a MiSeq V2 600 cycle kit (Illumina). The resulting data was then analyzed as described in Fox et al. (2019) . Phylogenetic trees were produced using the Maximum likelihood algorithm and 500 bootstraps in MEGA 7 (Kumar et al., 2016) . Pairwise identities were calculated using the same software.
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