strand specific reagent  (New England Biolabs)

Journal: PLoS Pathogens

Article Title: Nuclear-cytoplasmic compartmentalization of the herpes simplex virus 1 infected cell transcriptome is co-ordinated by the viral endoribonuclease vhs and cofactors to facilitate the translation of late proteins

doi: 10.1371/journal.ppat.1007331

Figure Lengend Snippet: HFFF cells were left uninfected, or infected with HSV1 (s17) at a multiplicity of 2. At 4 or 12 h.p.i., total RNA was purified and used for library preparation followed by sequencing. A total of 5 biological replicates were sequenced for each condition. (A) The proportion of reads mapped in each condition to either the human (blue) or HSV1 (red) transcriptome. (B) Differential expression analysis of cell and virus transcripts was conducted using EdgeR as described in Methods. Differences in the number of reads mapped to cell (black circles) and virus (green circles) transcripts were plotted as scatter plots comparing results at 4 and 12 hours to uninfected cells. The red dashed line is the line of identity (1:1) representing no change in the transcript levels between each condition. The 12 h results are also represented in a volcano plot indicating the high level of significance for the detected changes (right hand panel, where the red dashed line denotes a p-value of 0.05). (C) The reads obtained for the virus transcriptome were mapped to the virus genome for 4 (red) and 12 (green) hours. Numbers in parentheses represent maximum read counts per million obtained in each condition. The location of the TK, ICP27 and gD genes are indicated by arrows. (D) HFFF cells grown in slide chambers were infected with HSV1 (s17) at a multiplicity of 2 fixed at 4 or 12 h, and subjected to multiplex mRNA FISH with probes to genes representing IE (ICP27 in cyan), E (TK in red) and late (gD in green) transcripts. Nuclei were counterstained with DAPI (blue). Scale bar = 20 μm.

Article Snippet: Illumina RNA-Seq sequence libraries were constructed using the Strand-Specific RNA reagent kit (Agilent Technologies, G9691A), according to manufacturer’s instructions (Protocol Version E0, March 2017).

Techniques: Infection, Purification, Sequencing, Expressing, Multiplex Assay

Journal: PLoS Pathogens

Article Title: Nuclear-cytoplasmic compartmentalization of the herpes simplex virus 1 infected cell transcriptome is co-ordinated by the viral endoribonuclease vhs and cofactors to facilitate the translation of late proteins

doi: 10.1371/journal.ppat.1007331

Figure Lengend Snippet: (A) Plaque formation of a spontaneous variant of the Δ22 virus (Δ22*) in comparison to Wt and Δ22 on HFFF cells. (B) Line drawing of the HSV-1 vhs open reading frame denoting the 4 conserved boxes (I to IV) and the VP16 binding domain. Sequencing of the vhs gene from the Δ22* genome revealed a single amino acid change in the second conserved box of the protein (A95T). (C) HFFF cells were infected with Wt (s17), Δ22, Δvhs or Δ22* viruses at a multiplicity of 2, were metabolically labelled with [35S]-methionine 15 hours after infection. Cells were lysed and analysed by SDS-PAGE and autoradiography. (D) HFFF cells infected with Wt (s17), Δ22, Δvhs or Δ22* viruses at a multiplicity of 2 were harvested for total RNA at 16 hours and analysed by qRT-PCR of the indicated virus transcripts. Results are represented as Log 2 fold change to Wt (ΔΔCT), and the mean ± standard error for n = 3 is shown. Transcripts are colour-coded as blue (immediate-early) red (early), green (late) and pink (true late). (E) & (F) HFFF cells infected with Δ22 or Δ22* viruses at a multiplicity of 2 were fixed at 16 hours and subjected to PABP immunofluorescence in green (E) or multiplex mRNA FISH with probe specific for the late transcript gC in red (F). Nuclei were counterstained with DAPI (blue). Scale bar = 20 μm.

Article Snippet: Illumina RNA-Seq sequence libraries were constructed using the Strand-Specific RNA reagent kit (Agilent Technologies, G9691A), according to manufacturer’s instructions (Protocol Version E0, March 2017).

Techniques: Variant Assay, Binding Assay, Sequencing, Infection, Metabolic Labelling, SDS Page, Autoradiography, Quantitative RT-PCR, Immunofluorescence, Multiplex Assay