GSM8045429: HS2 H1N1 infected, then SARS-CoV-2 infected 48 hours later, Day 2, animal 1; Mesocricetus auratus; RNA-Seq
RNA was extracted (RNeasy Mini Kit; Qiagen) and potential DNA contamination were removed with 30 minute incubation in RNase-free DNase (Qiagen). Total RNA quality was determined by estimating the A260/A280 and A260/A230 ratios by nanodrop.   RNA integrity was determined by running an Agilent Bioanalyzer gel, with samples with values of 5 or greater used for library preparation. Using the Kapa RNA HyperPrep Kit with RiboErase (KR1351), rRNA was depleted starting from 25-1000ng of total RNA by hybridization of rRNA to complementary DNA oligonucleotides, followed by treatment with RNase H and DNase to remove rRNA duplexed to DNA.  Samples were then fragmented using heat and magnesium. 1st strand synthesis was performed using random priming.  2nd strand synthesis incorporated dUTPs into the 2nd strand cDNA.  Adapters were then ligated and the library amplified. Indexed libraries were quantified by qRT-PCR using a commercially available kit (KAPA Biosystems, Roche) and insert size distribution determined with the LabChip GX or Agilent Bioanalyzer. Samples with a yield of ?0.5 ng/ul were used for sequencing.   Sample concentrations were normalized to 1.2 nM and loaded onto an Illumina NovaSeq flow cell at a concentration that yielded at least 30 million passing filter clusters per sample. Samples were sequenced using 150bp paired-end sequencing on an Illumina NovaSeq according to Illumina protocols