The HSV-1 ICP22 protein selectively impairs histone repositioning upon Pol II transcription downstream of genes (RNA-seq II)

Part 1: Human telomerase reverse transcriptase (hTERT)-Immortalized cell line (T-HF) inducibly expressing ICP22 or ICP27 (doxycycline-induced) were analyzed for gene expression and read-through transcription by RNA-seq (total RNA). This experiment was performed in parallel to the corresponding OMNI-ATACseq libraries in replicate wells. ICP22 or ICP27 expression was induced for 24 h prior to other treatments or harvest, respectively. ICP22 expressing cells were additionally treated with KCl (200 mM) or control for 2 h prior to harvest. Part 2: Human telomerase reverse transcriptase (hTERT)-Immortalized cell line (T-HF) doxycycline-inducibly expressing an artificial miRNA against SSRP1 (two separate cell lines expressing different amiRNAs) were mock, HSV-1 strain F wild type or US1 deletion mutant infected to analyze gene expression and read-through transcription. This experiment was performed in parallel to the corresponding OMNI-ATACseq libraries in replicate wells. Expression of amiRNAs was induced for 72 h prior to infection. Cells were inoculated for 1 h and then incubated for another 8 h. The experiment was conducted with PAA (350 ug/ml) treatment, which was added after the inoculum was removed, and doxycycline where indicated. Part 1: ICP22 or ICP27 expression in T-HFs was induced with 5 ug/ml doxycycline for 24 h prior to other treatments or harvest, respectively. ICP22 expressing cells were additionally treated with KCl (200 mM) or control for 2 h prior to harvest. OMNI-ATACseq libraries were prepared with 50000 cells/sample as described by Corces et al. (2017). Two biological replicates were prepared. Part 2: Human telomerase reverse transcriptase (hTERT)-Immortalized cell line (T-HF) doxycycline-inducibly expressing an artificial miRNA against SSRP1 (two separate cell lines expressing different amiRNAs) were mock, HSV-1 strain F wild type (MOI 10) or US1 deletion mutant (MOI 10) infected to analyze gene expression and read-through transcription. This experiment was performed as a control in parallel to the corresponding OMNI-ATACseq libraries in replicate wells. Expression of amiRNAs was induced with 5 ug/ml doxycycline for 72 h prior to infection. Cells were inoculated for 1 h with MOI 10 and then incubated for another 8 h (inoculum was replaced with fresh medium). The experiment was conducted with PAA (350 ug/ml) treatment, which was added after the inoculum was removed, and 5 ug/ml doxycycline where indicated. Each amiRNA represents a separate biological replicate. For both parts the samples were harvested in TRIZOL and processed by standard methods. RNA was depleted of rRNA and libraries were prepared using the Truseq stranded library prep kit.