Journal: bioRxiv

Article Title: Interferon induced circRNAs escape herpesvirus host shutoff and suppress lytic infection

doi: 10.1101/2023.09.07.556698

Figure Lengend Snippet: A) Bulk RNA-Seq analyzed using CLEAR (CircExplorer3) from HSV-1 (MRC-5 infected with KOS MOI 10 for 12 hours, n=4), KSHV (iSLK-BAC16 reactivated with Dox/NaB for 3 days, n=4), and MHV68 (3T3 infected with H2B-YFP MOI 5 for 18 hours, n=2) infection. Graphs are limited to genes where raw BSJ and forward splice junction (FSJ) counts were >1 across all biological replicates. The average Log 2 FC Infected/Uninfected (HSV-1, MHV68) or Induced/Uninduced (KSHV) was plotted for linearFPB and circFPB, with each dot being a distinct gene. B) HEK-293 cells were transfected for 24 hours with plasmid vectors expressing GFP or viral endonucleases (vhs, BGLF4, SOX, muSOX). RNA was collected and assessed after reverse transcription using qPCR to quantitate mRNAs and noncoding RNAs (ncRNAs). Data is plotted as relative expression (ddCt) using 18S rRNA as the reference gene, and relative to a paired GFP transfected sample. Data points are biological replicates, column bars are the average, and error bars are standard deviation.

Article Snippet: RNA was rRNA depleted and directional cDNA libraries were generated using either Stranded Total RNA Prep with Ribo-Zero Plus (Illumina # 20040525) or TruSeq Stranded Total RNA Ribo-Zero Gold (Illumina #RS- 122-2303).

Techniques: RNA Sequencing, Infection, Transfection, Plasmid Preparation, Expressing, Reverse Transcription, Standard Deviation

Journal: bioRxiv

Article Title: Interferon induced circRNAs escape herpesvirus host shutoff and suppress lytic infection

doi: 10.1101/2023.09.07.556698

Figure Lengend Snippet: A-F) circRELL1 was depleted in MRC-5 cells using siRNAs for 48 hours and subsequently infected with HSV-1 strain KOS at MOI of 0.1 or 10 PFU/cell. Data is relative to a paired Non-Targeting Control siRNA (NTC). G-H) MRC-5 cells were infected with a lentivirus expressing circRELL1 for 48 hours and subsequently infected with HSV-1 strain KOS at MOI of 10 for 12 hours. Data is relative to a control lentivirus expressing circGFP. A, D, G) RNA was collected from the cell fraction and reverse transcribed. qPCR data is plotted as relative expression (ddCt) using 18S rRNA as the reference gene. B, E) DNA was isolated from the cell fraction and assessed by qPCR for viral and host genome copies. C, F, H) Supernatant was collected at 12 hpi and assessed by plaque assay. Data points are biological replicates, column bars are the average, and error bars are standard deviation. Paired two-tailed t-tests were performed and any p-value <0.1 are shown.

Article Snippet: RNA was rRNA depleted and directional cDNA libraries were generated using either Stranded Total RNA Prep with Ribo-Zero Plus (Illumina # 20040525) or TruSeq Stranded Total RNA Ribo-Zero Gold (Illumina #RS- 122-2303).

Techniques: Infection, Control, Expressing, Reverse Transcription, Isolation, Plaque Assay, Standard Deviation, Two Tailed Test