RNA extraction from cell pellets was performed by MagnaNAPpure LC 2.0 System using the MagNAPure RNA isolation kit high performance (Roche), according to the manufacturers’ instructions. To obtain a rough estimate of genomic viral RNA at each time point, RNA preparations (two replicates per condition) were merged into a single RNA pool per condition, then tested by RT–qPCR on a CFX96 Touch system (Bio-Rad Laboratories) with ANDiS FAST SARS-CoV-2 RT–qPCR Detection Kit (3D Biomedicine Science & Technology Co), targeting the protein gene regions N, E, and Orf1ab. RT–qPCR were used to estimate the genomic viral RNA content in each RNA pool on the basis of cycle threshold (Ct) values; samples showing a Ct value ≤ 30 were retrotranscribed and processed for NGS process.
Reverse transcription was performed as described elsewhere (Marcolungo et al, 2021 (link)). Two cDNA preparations were generated from each condition, and amplified using the ARTIC Primers v.1.3 (Marcolungo et al, 2021 (link)). PCR products were cleaned up using 1× AMPure XP beads (Beckman Coulter) and eluted in 15 μl of water. Amplicon libraries, prepared using the KAPA Hyper prep kit (Roche), were analyzed on the 4,150 TapeStation System (Agilent Technologies) and quantified using the Qubit dsDNA HS Assay kit (Thermo Fisher Scientific). Libraries were sequenced on Illumina MiSeq in 250-bp paired-end.
Reverse transcription was performed as described elsewhere (Marcolungo et al, 2021 (link)). Two cDNA preparations were generated from each condition, and amplified using the ARTIC Primers v.1.3 (Marcolungo et al, 2021 (link)). PCR products were cleaned up using 1× AMPure XP beads (Beckman Coulter) and eluted in 15 μl of water. Amplicon libraries, prepared using the KAPA Hyper prep kit (Roche), were analyzed on the 4,150 TapeStation System (Agilent Technologies) and quantified using the Qubit dsDNA HS Assay kit (Thermo Fisher Scientific). Libraries were sequenced on Illumina MiSeq in 250-bp paired-end.
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