DNA extracted from each S. pyogenes isolate (50-ng/sample) was prepared for sequencing with the use of the Nextera XT DNA Sample Prep Kit (Illumina). Clean up was performed using the AMPure XP PCR purification beads (Agencourt, Brea, CA, USA). The resulting individual DNA libraries with fragment sizes ranging from 500–1000 bp were quantified by quantitative PCR on a CFX96 (Bio-Rad, USA) in triplicate at two concentrations, 1:1000 and 1:2000, using the Kapa library quantification kit (Kapa Biosystems, Woburn, MA, USA). Based on the individual library concentrations, equimolar pools of the indexed libraries were prepared at a concentration of at least 1 nM using 10 mM Tris-HCl (pH 8.0) and 0.05% Tween 20. Pooled paired-end libraries were sequenced to a read length of at least 250 bp.
Ampure xp pcr purification beads
Manufactured by Beckman Coulter
Sourced in United States, United Kingdom
AMPure XP PCR purification beads are a solid-phase reversible immobilization (SPRI) technology used for the purification of PCR and sequencing reactions. The beads selectively bind DNA fragments based on their size, allowing for the removal of unwanted components such as primers, nucleotides, and salts from the reaction mixture.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Kapa hifi hotstart, by Roche (2 mentions)
Bcl2fastq v2.17.1.14 conversion software, by Illumina (2 mentions)
Nextera xt dna library prep kit, by Illumina (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Nextera xt dna sample prep kit, by Illumina (1 mentions)
Kapa library quantification kit, by Roche (1 mentions)
Cfx96, by Bio-Rad (1 mentions)
Miseq reagent kit v3, by Illumina (1 mentions)
Miseq system, by Illumina (1 mentions)
Agilent 2100, by Agilent Technologies (1 mentions)
Multiplex oligos for sequencing, by Illumina (1 mentions)
Miseq reagent kit version 2, by Illumina (1 mentions)
Nebnext ultra rna library prep kit, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Cas9 2a gfp, by Addgene (1 mentions)
Mobio powerplant dna isolation kit, by Qiagen (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
8 protocols using ampure xp pcr purification beads
1
Whole Genome Sequencing of Streptococcus pyogenes
2
Amplicon Library Sequencing on Illumina MiSeq
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An equal volume of each pooled amplicon library was independently cleaned and concentrated using Agencourt AMPure XP PCR purification beads according to manufacturer’s instructions. The two libraries (1-step and 2-step PCR) were subsequently spiked with 15% PhiX control DNA and sequenced in independent runs by the Center for Medicine and the Microbiome at the University of Pittsburgh on an Illumina MiSeq System with custom sequencing primers and a 2 × 300 bp MiSeq Reagent Kit v3 (Supplemental table 5 ). Sequences were demultiplexed and converted from base call files to FASTQ files using MiSeq Reporter software (v.2.6.2.3). FASTQ files for both index reads (i7 and i5) were generated by specifying the CreateFastqForIndexReads setting in the MiSeq Reporter.exe.config file. Raw reads are available from the Sequence Read Archive (BioProject PRJNA638427).
3
16S rRNA Gut Microbiome Analysis
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The genomic DNA from the cecal contents was amplified using a Taq DNA Polymerase kit according to the manufacturer's guidelines. The quality and quantity of genomic DNA were assessed by NanoDrop 2000) (Thermo, Waltham, USA). The 16S V3–V4 region of each sample was amplified using a MiSeq Reagent Kit v3, and purified using Agencourt AMPure XP PCR purification beads. The amplification primers were 5′-CCTACGGGNGGCWGCAG-3′ (forward) and 5′-GACTACHVGGGTATCTAATCC-3′ (reverse), and the quality of the products was assessed using an Agilent 2100 bioanalyzer (Agilent Technologies, CA, USA). The library was sequenced using a two-terminal sequencing strategy on the Miseq platform.
To improve the accuracy of the data, quality control was performed using TrimGalore, Mothur, and Usearch software. Sequences with ≥97% similarity were assigned to the same operational taxonomic units (OTUs). The OTUs were identified using Mothur software and the RDP database (http://rdp.cme.msu.edu/index.jsp ). Community bar-plot, alpha and beta diversity analyses, heat mapping, principal components analysis (PCoA), and species identification were performed using the Mothur and R software packages (http://www.R-project.org ), and linear discriminant analysis effect size (Lefse) was performed using Lefse (https://galaxyproject.org ).
To improve the accuracy of the data, quality control was performed using TrimGalore, Mothur, and Usearch software. Sequences with ≥97% similarity were assigned to the same operational taxonomic units (OTUs). The OTUs were identified using Mothur software and the RDP database (
4
mRNA Library Preparation for Illumina Sequencing
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Library preparation was performed on the selected mRNA using NEBNext Ultra RNA Library Prep Kit and Multiplex Oligos for Illumina Sequencing (New England Biolabs). Incubation and PCR steps were carried out by Veriti Thermocycler (Applied Biosystems/Life). During and after the protocol, DNA was purified using Agencourt AMPure XP PCR Purification Beads. Size selection of adapter-ligated DNA was performed immediately prior to final library amplification. This step allowed us to optimize fragment-size distribution for sequencing. Size selection was performed according to NEBNext Ultra Protocol, Version 2.0. Final PCR amplification consisted of 12 cycles. Libraries were then quantified and assessed for quality using an Agilent 2100 Bioanalyzer. Samples were sequenced using the MiSeq Version 2 Reagent Kit on the Illumina MiSeq platform. Samples were prepared according to manufacturer’s protocol (revision B). Each sample was sequenced with a single kit with 151-nucleotide paired-end reads.
5
Next-Generation Sequencing Library Preparation
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DNA amplicons for NGS were generated by PCR using KAPA HiFi HotStart (Roche), according to manufacturer’s instructions, using primers with overhangs compatible with Nextera XT indexing (IDT, Supplementary Table S2 ). Following validation of the quality of PCR products by gel electrophoresis, the PCR products were isolated using an AMPure XP PCR purification beads (Beckman Coulter). Indexed amplicons were then generated using a Nextera XT DNA Library Prep Kit (Illumina) quantitated, and pooled. Libraries were sequenced with a MiSeq Nano Flow Cell for 251 cycles from each end of the fragment using a MiSeq Reagent Kit v2 (500-cycles). FASTQ files were created and demultiplexed using bcl2fastq v2.17.1.14 Conversion Software (Illumina). Deep sequencing was performed by the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign.
6
CRISPR-Mediated TP53 Knockout in hESCs and RKO Cells
7
Fungal Community Profiling of Salt Marsh Plant Roots
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DNA was extracted from 0.05 g of homogenised dry roots using MoBio PowerPlant DNA isolation kit following the manufacturer’s instructions (MoBio Laboratories Inc., Carlsbad, CA, USA). In order to quantify the diversity and composition of fungal communities associated with salt marsh plant roots, the internal transcribed spacer (ITS1) region was PCR-amplified from homogenised roots with the primers ITS1f and ITS2 [48 , 49 (link)]. These primers target all major phyla of fungi (Ascomycota, Basidiomycota, Chytridiomycota, Glomeromycota, Mucoromycotina and Zygomycota), but exclude the unicellular animal–parasite group Microsporidia. PCR products were bead purified using Agencourt AMPure XP PCR Purification beads (Beckman Coulter Ltd, High Wycombe, UK), before sample-specific Nextera XT indices were added to amplicons with a short (8) cycle PCR. After pooling samples in equimolar concentrations, sequencing was conducted on an Illumina HiSeq 2500 in rapid run mode (providing 2 × 300 bp sequences) at The Earlham Institute (formerly The Genome Analysis Centre, Norwich Research Park, Norwich, NR4 7UH, UK). A more detailed description of molecular workflows is available in the Supplementary Information (Methods S1) .
8
Nextera XT Library Preparation for NGS
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DNA amplicons for NGS were generated by PCR using KAPA HiFi HotStart (Roche), according to the manufacturer’s instructions, using primers with overhangs compatible with Nextera XT indexing (IDT, Supplementary Table S2 ). Following validation of the quality of PCR products by gel electrophoresis, the PCR products were isolated using AMPure XP PCR purification beads (Beckman Coulter). Indexed amplicons were then generated using a Nextera XT DNA Library Prep Kit (Illumina), quantitated, and pooled. Libraries were sequenced with a MiSeq Nano flow cell for 251 cycles from each end of the fragment using a MiSeq Reagent Kit v2 (500 cycles). FASTQ files were created and demultiplexed using bcl2fastq v2.17.1.14 Conversion Software (Illumina). Deep sequencing was performed by the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign.
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