RNA-Seq of Murine Fibroblasts Infected by Lab Strain wt (3 hpi)
Total RNA was isolated from infected L929 cells at 1 hour and 3 hours post infection (hpi) using the TRIZol Plus RNA Isolation Kit (Life Technologies) according to the manufacturers directions. To ensure that the highest quality RNA is used in this analysis, 4 biological replicates of each condition were isolated. RNA was quantitated via nanodrop and bioanalyzer. Libraries were prepared using the Illumina TruSeq strand specific mRNA sample preparation system (Illumina).Briefly, mRNA was extracted from total RNA using polyA selection, followed by RNA fragmentation. Strand specific library was constructed by first-strand cDNA synthesis using random primers, sample cleanup and second-strand synthesis using DNA Polymerase I and RNase H. A single 'A' base was added to the cDNA fragments followed by ligation of the adapters. Final cDNA library was achieved by further purification and enrichment with PCR, quality checked using Agilent 4200 TapeStation. The libraries were sequenced (Single end 100bp reads) using the Illumina HiSeq4000, with 10 samples per lane, and final of over 20 million reads per sample.