Alu DNA sequences were from the GrCh37 (hg19) assembly. We designed unedited Alu DNA templates and changed A nucleotides edited in HC but not COV-S to G nucleotides as a mimic of A-to-I editing. A SP6 promoter was added to the 5’ end and synthetic DNAs were obtained from IDT (32 ). RNA transcription was performed using Megascript SP6 (Invitrogen). THP-1 reporter cell lines (Invivogen) contained stably integrated luciferase genes under the control of either an IFN-stimulated response element (ISRE) or NF-kB response element. Transfections were performed using Lipofectamine® RNAiMAX (ThermoFisher Scientific) (33 ). Luciferase activity was determined after 24 hr. using luciferin substrate (Invivogen) and light emission measured with a TD20/20 luminometer. Gene expression measurements were performed as previously described (19 (link), 20 (link)).
Protocol detail
Alu DNA Editing and Reporter Assay
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Dna sequences
Dnas
Gene expression
Genes control
Light
Lipofectamine
Luciferase
Luciferin
Nf kb
Nucleotides
Response element
Thp 1 cell lines
Transcription
Transfections
Corresponding Organization :
Other organizations : Vanderbilt University, Vanderbilt University Medical Center
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Variable analysis
independent variables
- Edited Alu DNA templates (with A nucleotides changed to G nucleotides as a mimic of A-to-I editing)
- Luciferase activity (after 24 hr. transfection)
- Unedited Alu DNA templates
- Positive control: THP-1 reporter cell lines containing stably integrated luciferase genes under the control of either an IFN-stimulated response element (ISRE) or NF-kB response element
- Negative control: Not explicitly mentioned
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