TruSeq Stranded mRNA Library Prep Kit to select poly(A) RNA, followed by the remaining modules of an Illumina TruSeq Stranded mRNA Library Prep Kit. 2.10 RNA sequencing and analysis. The sequencing libraries were analyzed on an Illumina MiSeq, collecting single-end data sets of 75 nucleotides. The sequence of HCMV strain AD169 in GenBank accession BK000394 was used to align read data and specify sequences representing exon-exon junctions. Whole genome transcriptome profiles were visualized by using Artemis (), and reads representing exon-exon junctions were enumerated by using the Unix grep program. 3. RESULTS 3.1 High throughput screening of a compound library. We screened the Gray Kinase Inhibitor library of 187 compounds S1) for anti-HCMV activity using a methodology based on our previous siRNA screening experiments (). Briefly, HFF cells were treated with each compound from the collection and infected with HCMV strain AD169. Cells were then stained with antibodies to detect the HCMV antigen pp28. As pp28 is expressed late in infection, detection of pp28 allowed our screen to identify compounds affecting all stages of HCMV replication from entry of virus into the cell to late gene expression. The total number of cells and the number of infected cells were enumerated using automated microscopy. In wells where the mean number of
TruSeq Stranded mRNA Library Prep Kit to select poly(A) RNA, followed by the remaining modules of an Illumina TruSeq Stranded mRNA Library Prep Kit. 2.10 RNA sequencing and analysis. The sequencing libraries were analyzed on an Illumina MiSeq, collecting single-end data sets of 75 nucleotides. The sequence of HCMV strain AD169 in GenBank accession BK000394 was used to align read data and specify sequences representing exon-exon junctions. Whole genome transcriptome profiles were visualized by using Artemis (), and reads representing exon-exon junctions were enumerated by using the Unix grep program. 3. RESULTS 3.1 High throughput screening of a compound library. We screened the Gray Kinase Inhibitor library of 187 compounds S1) for anti-HCMV activity using a methodology based on our previous siRNA screening experiments (). Briefly, HFF cells were treated with each compound from the collection and infected with HCMV strain AD169. Cells were then stained with antibodies to detect the HCMV antigen pp28. As pp28 is expressed late in infection, detection of pp28 allowed our screen to identify compounds affecting all stages of HCMV replication from entry of virus into the cell to late gene expression. The total number of cells and the number of infected cells were enumerated using automated microscopy. In wells where the mean number of
blsGrayCollectionScreen%2DFINAL%20R1.docx