Qubit rna hs

Total DNA and RNA were extracted from sorted, pure CD33⁺HLA-DR^– and CD33⁺HLA-DR⁺ myeloid cell populations, isolated from ten healthy donors, using Norgen RNA/DNA/protein purification Plus Micro Kit (Norgen Bioteck Corporation, Ontario, Canada) according to the manufacture’s protocol. RNA was then amplified using 5X MessageAmp™ II aRNA Amplification Kit (Invitrogen). RNA concentrations were determined by Qubit RNA HS and Broad Range Assay Kits, respectively (Invitrogen).

Total RNA was isolated from infected plant samples using the EZNA Plant kit (Omega) according to the manufacturer's recommendations, including the on‐column DNase treatment, which was carried out using Turbo DNase I (Invitrogen). RNA was checked for quality and quantity using Caliper LabChip GX (Perkin Elmer) and Qubit RNA HS (Thermo Fisher Scientific) analysis, respectively. To enrich RNA samples for bacterial messenger RNA, multiple depletion reagents were used, including oligo(dT) beads to deplete eukaryotic polyadenylated mRNA, RiboZero Plant rRNA removal beads to deplete host rRNA, and RiboZero bacterial rRNA removal beads to remove prokaryotic rRNA (Illumina). The remaining RNA was used for library preparation using an Illumina TruSeq Stranded Total RNA Library Preparation Kit (Illumina) on a Sciclone G3 robot following the manufacturer's recommendations (Perkin Elmer). Library quality was assessed using Caliper LabChipGX HS DNA (Perkin Elmer) and Qubit dsDNA HS (Thermo Fisher Scientific) assays. Samples were pooled and run on three lanes using the Illumina HiSeq4000 platform with single‐end 50 bp format. Sequencing was conducted at the Michigan State University Research Technology Support Facility. Base calling was conducted by Illumina Real Time Analysis v. 2.7.6.

3′ RNA-seq was conducted by the Molecular Genomics Core (PMCC, Melbourne). The total RNA quantity was measured using Qubit RNA HS (Thermo Fisher Scientific). 500 ng total RNA was used for library preparation according to standard protocols (QuantSeq 3′ mRNA-seq FWD, Lexogen). Indexed libraries were pooled and sequenced on a NextSeq500 (Illumina). 5–15 million single-end 75 bp reads were generated per sample. Sequenced reads were trimmed and aligned to hg38 genome via Cutadapt and HISAT2. Gene counts were obtained from featureCounts. Differential expression was performed using Limma. Genes were considered as DEGs if the absolute log-fold change was >2 and p value <0.05. All analysis packages were operated within the Galaxy suite environment. The functional enrichment analysis of annotated terms from GO was performed with the online tool DAVID using the human genome as the background. The gene-gene interaction analysis was completed using the GeneMania app in Cytoscape 3.