GSM6381557: WT, 8 hpi, Rep1; Homo sapiens; RNA-Seq
Three biological replicates per time point were obtained by independent infections. Cells were harvested at the indicated times post-infection by trypsinization, washed with PBS and centrifuged at 5000 xg for 2 minutes. The resulting pellets were lysed with TRIzol (Thermo Fisher 15-596-026) and total RNA was harvested according to manufacturer's instructions. RNAs were then treated with DNase (Qiagen 79254) on RNeasy columns (Qiagen 74104) per manufacturer's instructions. To improve RNA quality, RNAs were precipitated with 3 volumes ice-cold 96% ethanol (Fisher BP8202500), 1 volume 3 M sodium acetate pH 5.5 (Fisher BP333-500) and 1 µL glycogen (Thermo Fischer R055) overnight at -80 °C. Precipitated RNAs were pelleted at 15,000 xg and 4 °C for 30 min, washed with ice-cold 75% ethanol and spun as above for 10 min. Ethanol was removed and RNAs were resuspended in nuclease-free water in Lo-Bind microcentrifuge tubes (Eppendorf 022-43-102-1). RNA concentrations were quantified by Nanodrop and their integrity confirmed using the 4200 Tapestation Bioanalyzer system (Agilent). 500 ng of total RNA with an RNA Integrity Number (RIN) greater than 9.5 were used to prepare sequencing libraries with the Illumina TruSeq Stranded mRNA Library Prep Kit (Illumina). Library concentrations were measured with Invitrogen Qubit dsDNA HS Assay Kit then analyzed with Agilent High Sensitivity D5000 ScreenTape System and pooled. Libraries were sequenced with 100-bp paired-end reads on an Illumina NovaSeq 6000 SP sequencer at the Fred Hutch Genomics Core Facility.