Journal: Cell Reports Medicine

Article Title: Haploinsufficiency of NFKBIA reshapes the epigenome antipodal to the IDH mutation and imparts disease fate in diffuse gliomas

doi: 10.1016/j.xcrm.2023.101082

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Article Snippet: For near-complete knockdown of NFKBIA , G418-resistant primary human astrocytes transduced to stably express wildtype IDH1 or mutant IDH1- ( R132H ) were transfected with Accell human NFKBIA small interfering (si)RNA or non-targeting control siRNA (Dharmacon), at 20nM concentration using lipofectamine 2000 reagent (Invitrogen) at 1:1 ratio for 48 h. For complete clustered regularly interspaced short palindromic repeats (CRISPR) knockout of NFKBIA , astrocytes were transfected with IκBα Double Nickase Plasmid (h) (sc-400034-NIC, Santa Cruz)—consisting of a pair of plasmids each encoding a D10A mutated Cas9 nuclease and a target-specific 20-nucleotide (nt) guide RNA (gRNA)—using plasmid transfection medium (sc-108062) and Ultra-Cruz transfection reagent (sc-395739) at 1:1.5 ratio and were incubated for 72 h. After incubation, cells were screened for GFP-positivity to select for successfully transfected cells.

Techniques: Plasmid Preparation, Recombinant, Transfection, Activation Assay, Methylation, Marker, DNA Methylation Assay, Sequencing, Empire Assay, Software