Nebnext globin rrna depletion kit

A volume of 300 to 500 μL of anticoagulated whole blood will be used to extract total RNA from leukocytes using the RiboPure RNA Purification Kit (Thermo Fisher Scientific). Infected tissue biopsies (10-30 mg) will be disrupted and homogenized with TissueLyser (Qiagen), and total RNA of both human and bacterial origin will be isolated using the RNeasy Plus Mini Kit (Qiagen). We will use the NEBNext Globin & rRNA Depletion Kit (New England Biolabs Inc) for strand-specific messenger RNA (mRNA) purification using probes that are selective for globin mRNA, cytoplasmic ribosomal RNA (rRNA), and mitochondrial rRNA with human, mouse, and rat samples. In all biological samples, first- and second-strand copyDNA (cDNA) will be synthesized based on ligation adaptor techniques (NEBNext Globin & rRNA Depletion Kit [human, mouse, and rat]). Each step will be performed according to the manufacturer’s instructions and an internal standard operational procedure. The obtained cDNA library will then be sequenced with dual RNA sequencing with paired-end sequencing of 150 nucleotide fragments on the Illumina Novaseq6000 platform with a targeted sequencing read depth of 20 million reads.

RNA sequencing was performed on RNA extracted from the whole blood of the 96 patients with miRNA data generated. The extracted RNA samples (400 ng) were subject to globin and ribosomal RNA depletion using the NEBNext® Globin & rRNA Depletion Kit (as per manufacturer’s protocol; New England Biolabs). Preparation of cDNA libraries was subsequently performed using the NEBNext® Ultra II Library Prep Kit for Illumina (New England Biolabs). Libraries were checked for quality and quantified with a 2100 Bioanalyzer instrument, and then pooled into one lane of an S2 flow cell and 101-bp paired-end sequenced on a NovaSeq instrument (Illumina) in XP mode. The mRNA data is deposited in GEO under accession GSE220076.