GSM8945731: SARS-CoV-2 Wiltype replicate 4; Chlorocebus aethiops; RNA-Seq
Total cellular RNA was extracted via TRIzol purification for analysis (SARS-CoV-2). 300 ng to 1 µg of total cellular RNA from rMHV-infected L929 cells or Vero E6 cells infected with rSARS-CoV-2 WT, rSARS-CoV-2 DORF8, rSARS-CoV-2 DORF7a, or rSARS-CoV-2 ORF8 DG was used as a template for reverse transcription and (-) sgRNA library generation using the QuantSeq-Flex kit (Lexogen Inc., Greenland, NH, USA). Libraries were created according to the manufacturer's instructions. Briefly, primers (see below) which bind to nucleotides 26 to 48 of the SARS-CoV-2 or nucleotides 35 to 54 of the rMHV (-) sgRNA 3′ end leader regions, respectively, were used for first strand synthesis. The template RNA was degraded prior to second strand synthesis using reagents provided in the kit. Libraries were amplified via PCR using QuantSeq-Flex kit reagents. Libraries at a final concentration of 1-2 ng DNA/μL were sent to the Yale Center for Genomic Analysis (YCGA) for sequencing (SARS-CoV-2). 10 μg of total RNA isolated from rMHV-infected cells was treated with DNase I (NEB, Ipswich, MA) according to the manufacturer's instructions. Total RNA was isolated from the DNase I reaction via acid phenol:chloroform extraction and isopropanol precipitation. Briefly, equal parts of acid phenol and chloroform were added to the DNase I reaction. Tubes were placed on ice and centrifuged at ~12,000 x g at 4°C in a table-top centrifuge. The aqueous phase was transferred to a new tube and precipitated with 40 μL 3 M sodium acetate, 12 μL glycerol, and 1 mL of 100% isopropanol for 1 hour at -20°C. Tubes were centrifuged for 10 minutes at ~20,000 x g. The pellets were washed with 70% ethanol and resuspended in 20 μL of nuclease free H2O. RNA was incubated at 55°C for 15 minutes for homogenization. 1 µg of DNase I treated RNA was spiked with 50 ng of yeast RNA. Samples were submitted to Boston University (BU) Sequencing Core for poly(A) selection, library preparation, and sequencing (MHV).