Summary: RNA-seq was employed to investigate host responses to SARS-CoV-2 infection in the bronchoalveolar lavage fluid (BALF) and peripheral blood mononuclear cells (PBMC) samples of COVID-19 patients.
One microgram of total RNAs extracted from PBMCs (Ficoll preparation) were used as input. Messenger RNAs were purified using oligo-dTs covalently coupled magnetic beads. Then, the RNAs were fragmented into small pieces by heating. First strand of cDNA was synthesized in the presence of specific chemicals to ensure that only RNAs were used as template. Double strand cDNAs were purified with Agencourt AMPure XP beads after the reaction. DNA library was constructed through end-repair, adaptor-ligation and PCR amplification. The intermediate products were size-selected after the adaptor-ligation using two rounds of Agencourt AMPure XP beads. Qualified double strand DNA library was transformed into single-stranded circular DNA library through DNA-denaturation and circularization. DNA nanoballs (DNBs) were generated from single-stranded circular DNA using rolling circle amplification (RCA). The DNBs were qualified using Qubit 2.0. Qualified DNBs were loaded on the flow cell and sequenced.; Total RNAs were extracted from BALF samples and quantified by Qubit RNA HS Assay Kit. The library preparation was performed with Trio RNA-Seq kit with AnyDeplete probe to remove human ribosomal RNAs. The resulting libraries were subject to 150 bp pair-end sequencing with an Illumina Miseq platform.
Illumina NovaSeq 5000; Illumina MiSeq; BGISEQ-500
Transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in COVID-19 patients.