RNA seq of homo sapien, infected with Dengue virus
Nucleic acid isolation:RNA extraction from the blood samples was carried out using the Qiagen QIAamp RNA Blood Mini Kit, following the manufacturer's instructions with specific modifications. The incubation and centrifugation time during the erythrocyte lysis step were reduced to 5 minutes. Additionally, for enhanced RNA purification, a 2-3 minute incubation period was introduced during all washing steps. The purity of the isolated RNA was assessed using a NanoDrop Microvolume Spectrophotometer and confirmed by running it through an agarose gel. Subsequently, the RNA was stored at -80 C until thawed for RNA-Seq library preparation. Library preparation and sequencing:Libraries were prepared with a total of 250ng of RNA using the Illumina TruSeq Stranded Total RNA Library Prep Globin (Illumina, Cat. No. 20020612). Initial steps involved the depletion of globin mRNA and ribosomal RNA (both cytoplasmic and mitochondrial), as these two forms of abundant RNA are present in high levels in whole blood. Cleaved RNA fragments underwent first strand cDNA synthesis through reverse transcriptase and random primers. Subsequently, double-stranded cDNA was synthesized using DNA polymerase 1 and RNase H, followed by purification using AMPure XP (Beckman Coulter, A63881). Adenylation at the 3 blunt end of double-stranded cDNA was performed, and sequencing libraries were uniquely indexed and enriched through PCR amplification.Library quality was confirmed through size analysis on an Agilent 2100 Bioanalyzer, and library concentrations were determined using the Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kit (Thermo Fisher Scientific; catalog no. Q32854). Libraries were then diluted to 4nM and combined in an equimolar fashion, with 24 samples per library pool. Paired-end, 2151 read length sequencing was performed on a NextSeq 2000 platform (Illumina) at a final loading concentration of 650 pM.