GSM4656062: SARS-CoV-2 genomic RNA; Severe acute respiratory syndrome coronavirus 2; RNA-Seq
The cells were centrifuged at 1,000g for 5 min at 4 °C. The cells were washed twice by resuspending the pellet with 15 ml PBS with 30% β-mercaptoethanol and centrifugation to pellet then just once with 15 ml PBS. After washes, the pellet was resuspended in 1 ml Trizol (ThermoFisher Scientific) and RNA was extracted following the manufacturer's specifications. Total RNA was purified using RNA Clean and Concentrator -25 kit (Zymo). rRNA was subtracted from the RNA extracted from Vero cells using rRNA subtraction mix and RNase H (Lucigen). RNA was fragmented at 70 °C for 2.5 min. The ends were dephosphorylated and repaired with Shrimp Alkaline Phosphatase at 37 °C for 1 h. Linker was added with T4 RNA ligase at 22 °C for 18 h. RNA was reverse-transcribed with TGIRT-III (Ingex) at 60 °C for 1.5 h. RT product was gel-purified and size-selected for ~250 nt, circularized with CircLigase (Lucigen), and PCR-amplified with Phusion High-Fidelity DNA Polymease (NEB) for up to 16 cycles. The product was run on a gel to select for fragments ~300 nt and sequenced on an iSeq100 (Illumina).