RNA-seq of A20 mouse cells using a novel TETact activation of CD4

Stably silenced genes that display a high level of CpG dinucleotide methylation are refractory to the current generation of CRISPR dCas9-based activation systems. To counter this, we created an improved activation system by coupling the catalytic domain of DNA demethylating enzyme TET1 with transcriptional activators (TETact). To evaluate the specificity of our TETact system, we conducted RNA-seq on A20 cells containing the TETact-v3 system targeting the CD4 promoter, along with non-targeting control TETact-v3 cells as well as wildtype A20. A20 is a BALB/C B cell lymphoma cell line that does not normally express CD4. Non-transduced A20 showed a very similar gene expression profile with the control TETact-v3 cells. Importantly, comparing of TETact-v3 cells targeting CD4 promoter to with cells expressing non-targeting sgRNA revealed CD4 as the sole significantly upregulated gene. Expression of the other genes in the CD4-targeting samples correlated strongly with the control sample. Together these experiments reveal the exquisite specificity of the TETact system. Two biological replicates were prepared of wild-type A20 cells, A20 cells with CD4 activators and A20 cells with non-targeting activators. RNA-seq profiles were prepared of all six samples.