Dsdna hs assay kit

We PCR amplified V3-V4 hyper-variable region of 16S rRNA gene using Pro341F/Pro805R^{33 (link),34 (link)} or 341F/805R primer pair^{35 (link),36 (link)} and the 16S metagenomic sequencing library preparation was performed using standard Illumina protocol (https://support.illumina.com/downloads/16s_metagenomic_sequencing_library_preparation.html). The obtained amplicon libraries were purified using 1X AMpureXP beads and checked on Agilent DNA1000 chip using Bioanalyzer2100. The quantification of the purified library was performed in Qubit Fluorometer2.0 using Qubit dsDNA HS Assay kit and the sequencing of amplicons were performed on Illumina MiSeq (2 × 300 bp reads)/HiSeq 2500 using Rapid Run chemistry (2 × 250 bp reads).

Initially, a bacterial cell pellet was obtained by centrifugation of 1 mL sample for 5 min at 9000× g. DNA was extracted via the SPINeasy DNA Kit for Soil (MP Biomedicals, Eschwege, Germany), according to manufacturer’s instructions. Subsequently, DNA libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA) and IDT Unique Dual Indexes with total DNA input of 1 ng. Genomic DNA was fragmented using a proportional amount of Illumina Nextera XT fragmentation enzyme. Unique dual indexes were added to each sample followed by 12 cycles of PCR to construct libraries. DNA libraries were purified using AMpure magnetic Beads (Beckman Coulter, Brea, CA, USA), eluted in QIAGEN EB buffer, quantified using a Qubit 4 fluorometer and a Qubit dsDNA HS Assay Kit, and sequenced on an Illumina Nextseq 2000 platform 2 × 150 bp. Unassembled sequencing reads were converted to relative abundances (%) using the CosmosID-HUB Microbiome Platform (CosmosID Inc., Germantown, MD, USA) [66 (link),67 (link)].

Total RNA was submitted to the Centre for Genomic Research Facility, University of Liverpool, for RNA-Seq library preparation and sequencing. One microgram of DNA-free total RNA was selected for poly-A using NEBNext Poly (A) mRNA magnetic isolation module (New England Biolabs UK Ltd, UK). RNA-Seq libraries were prepared from the enriched RNA using the NEBNext Ultra Directional RNA library Prep kit for Illumina. Libraries were purified using AMPure XP beads. Each library was quantified using Qubit and the size distribution assessed using the Agilent 2100 Bioanalyser.
Libraries were pooled and assessed by a Qubit dsDNA HS assay kit and qPCR using the Illumina Library Quantification kit from Kapa on a Roche Light Cycler LC48011. Concentrations of template DNA were adjusted to 3nM and denatured with 0.1N NaOH. Denatured cDNA was further diluted to a final concentration of 300pM. Clustering of the DNA templates was performed using HiSeq 3000/4000 paired end cluster Kit with a cBot cluster generation system (Illumina, San Diego, USA) according to the manufacturer’s instructions. Libraries were sequenced on an Illumina HiSeq 4000 using sequencing by synthesis chemistry to generate 2 x 150bp paired end reads.