Sparq dna library prep kit

Before the library preparation, the DNA concentration of each sample was also assessed by Qubit Fluorometric Quantification (Thermo Fisher Scientific, San Jose, CA, United States). Ten nanograms of each Qubit quantified genomic DNA was sheared with a Covaris E220 instrument operating SonoLab v6.2.6 generating approximately 300 bp DNA fragments according to the manufacturer’s protocol. Between 10 and 100 ng of fragmented DNA was processed into Illumina compatible sequencing libraries using sparQ DNA Library Prep Kit (Quantabio, Beverly, MA, United States). Each library was barcoded with unique dual index sequences (NEXTFLEX^® Unique Dual Index Barcodes, BioO Scientific). Library size and amount were confirmed with a Bioanalyzer High Sensitivity DNA chip. Polymerase chain reaction primers and reagents included in the sparQ kit were used to perform PCR, and products were purified with AMPure XP beads. Equimolar libraries were pooled and subjected to Illumina NovaSeq 6000 sequencing at 2 × 150 bp (Illumina, San Diego, CA, United States). Shotgun whole metagenome sequencing was performed at the Genome Sciences and Bioinformatics Core at the Pennsylvania State University College of Medicine, Hershey, PA, United States. Illumina bcl2fastq (released version 2.20.0.422) was used to extract de-multiplexed sequencing reads.

5’ RACE-PCR was performed on 1 µg of total RNA isolated from Hyaloperonospora-infected Arabidopsis leaves pooled from equal amounts isolated at 4 and 7 dpi, using the 5’/3’ RACE Kit, 2nd Generation (Roche Diagnostics). After the first round of PCR, a gel fraction of the expected size was cut out and a nested PCR was carried out on the eluted DNA. Bands were cut out and DNA was eluted using GeneJet Gel Extraction Kit (Thermo Fisher Scientific). A library was constructed from the eluted PCR fragments using the sparQ DNA Library Prep Kit (Quantabio) and sequenced on an Illumina MiSeq platform.