Journal: PLoS Pathogens

Article Title: A comparative epigenome analysis of gammaherpesviruses suggests cis-acting sequence features as critical mediators of rapid polycomb recruitment

doi: 10.1371/journal.ppat.1007838

Figure Lengend Snippet: (A) H3K27-me3 ChIP-seq coverage across the MHV-68Δ50 genome in MLE12 cells sorted for GFP expression at day 5 post infection with MHV-68Δ50-kLANA (top) or MHV-68Δ50 (bottom). Due to the low titers of the MHV-68Δ50-kLANA virus, ChIP-seq was performed for both cultures using a low-cell ChIP protocol as described in the material and methods section. (B) Relative H3K27-me3 enrichment analysis for the data shown in panel A, performed as described in the legend to . Significance was calculated by 1way ANOVA testing (F = 640.7, df = 781) and is indicated by asterisks or ns (not significant). (C) Confirmatory ChIP-qPCR analysis of MHV-68Δ50-kLANA or MHV-68Δ50-infected MLE12 cells at 5 days post infection (n = 1, top panel), or after a 35 day period during which cells were repeatedly sorted to achieve a population of 100% GPF positive cells (n = 2, bottom panel). Data are represented as mean ± SEM.(D) Western blot analysis of mLANA and kLANA expression in cells infected with MHV-68Δ50-kLANA or MHV-68Δ50, respectively. Unspecific bands are indicated by asterisks.

Article Snippet: Cell fixation was performed using the fixation reagents and steps from the Low Cell Chip Kit (Active Motif; Cat#53084) as described in the user manual.

Techniques: ChIP-sequencing, Expressing, Infection, Virus, ChIP-qPCR, Western Blot

Journal: PLoS Pathogens

Article Title: A comparative epigenome analysis of gammaherpesviruses suggests cis-acting sequence features as critical mediators of rapid polycomb recruitment

doi: 10.1371/journal.ppat.1007838

Figure Lengend Snippet: (A) ChIP-qPCR analysis of H3K27-me3 (top), H3K4-me3 (center) or IgG (negative control, bottom) in splenocytes harvested from two mice (#1 and #2) that had been intranasally infected with wildtype MHV-68 for 17 days, using MHV-68 or endogenous control primers as indicated. Data are represented as mean ± SEM of three independent ChIP replicates performed with the isolated chromatin from each mouse. (B) H3K27-me3 ChIP-seq coverage across the MHV-68 genome in two pools of splenocytes isolated from a total of six mice that had been intranasally infected with MHV-68-H2BYFP for 17 days. Splenocytes were FACS sorted for YFP expression prior to analysis. Due do the low number of positive cells, YFP-positive cells from three mice were pooled to generate pools #1 and #2. Approximately 5000 cells were subjected to low cell ChIP-seq using an H3K27-me3 specific antibody. Hashed boxes above the MHV-68 map indicate the position of repetitive regions (left and right internal repeat regions, as well as terminal repeat sequences) which had to be masked since they do not allow unique read mapping. (C) Relative enrichment of H3K27-me3 at viral episomes in splenocyte pools #1 and #2 was assessed using the same statistical method as described in the legend to . (D) Normalized mean H3K27-m3 and input coverage from all MHV-68 ChIP-seq experiments performed in our study. Quantile normalized mean values and standard deviation (colored area) of H3K27-me3 and input tracks were generated from all MHV-68-specific coverage tracks as given in (ChIP MHV-68).

Article Snippet: Cell fixation was performed using the fixation reagents and steps from the Low Cell Chip Kit (Active Motif; Cat#53084) as described in the user manual.

Techniques: ChIP-qPCR, Negative Control, Infection, Control, Isolation, ChIP-sequencing, Expressing, Standard Deviation, Generated