GSM8508077: Sample38_Passage1, COV-siRNA2, 20X; Severe acute respiratory syndrome coronavirus 2; OTHER
300 μL of cell supernatant was added to 900 μL of Trizol and stored at -80°C for further analysis. Trizol collected samples were subjected to RNA isolation using PureLink RNA Mini Kit with the incorporation of on-column PureLink DNase Treatment. Reverse transcription reactions were performed with 6 μL of purified RNA and random hexamer primers using the NEB ProtoScript II First Strand cDNA Synthesis kit. The resulting cDNA was used as a template for PCR amplification of the siRNA binding sites, two primer pairs per site, using KapaBiosystems polymerase (KAPA HiFi HotStart ReadyMix) with primers, 5'-acgctgcttctggtaatc-3' plus 5'-agaaacccttagacacagc-3', and 5'-ctagataaacgcactacgtg-3' and 5'-cctgagcaaagaagaagtg-3' for the COV-siRNA1 binding site, and  5'-agatgccttcaaactcaac-3' plus 5'-ttctactctgagttgttgc-3', and 5'-ttggtggcaaaccttgtatc-3' plus 5'-tgtgtaactggacacattg-3' for the COV-siRNA2 binding site. Each PCR with a primer pair was carried out independently, then PCR products from each treatment were pooled for purification and subsequent library preparation. Amplification conditions included an initial 3 min at 95°C, followed by 24 cycles with 20 sec at 98°C, 15 sec at 60°C and 72°C for 15 sec, with a final 1 min at 72°C. Pooled PCR products were purified using AMPure XP beads. The size of the amplicon was confirmed by analyzing 2 μL of PCR products using the Agilent D1000 ScreenTape System. Products were quantified by analyzing 1 μL with the Quant-iT dsDNA High-Sensitivity Assay Kit. Fifty ng of purified PCR product was used as input for library construction using the NEBNext Ultra II DNA Library Prep kit. NEBNext Multiplex Oligos for Illumina Dual Index Primer Set 1 was used for library construction, with a total of 4 PCR cycles. Library size was determined using the Agilent D1000 ScreenTape System and quantified with the Quant-iT dsDNA High-Sensitivity Assay Kit. Equal amounts of each library were pooled together for multiplexing and 'Protocol A: Standard Normalization Method' of the Illumina library preparation guide was used to prepare 8 pM final multiplexed libraries with 1% PhiX spike-in for sequencing. The MiSeq Reagent Kit v3 (600-cycle) was used for sequencing the libraries on the Illumina MiSeq platform, with 150 cycles for Read 1, 150 cycles for Read 2, 8 cycles for Index 1, and 8 cycles for Index 2. Illumina sequencing for COV-siRNA2 resistance studies was conducted by Genewiz/Azenta using an equivalent protocol.