GSE121757
Assessing the effect of SUPT4H1 RNAi on the transcription of a repeat-containing reporter construct
SUPT4H1 is a transcription elongation factor comprising part of the RNA polymerase II complex. Recent studies propose a selective role for SUPT4H1 in transcription of repeat-containing transcripts, the translated products of which are a hallmark of neurodegeneration in disorders such as Huntington's Disease and C9orf72-amyotrophic lateral sclerosis. To investigate the potential of SUPT4H1 as a therapeutic target in repeat-associated neurodegeneration, we engineered a reporter containing 71 hexanucleotide repeats (HNRs) encoding for pDi-amino acid repeats (DARPs) fused to EGFP. To approximate endogenous expression levels, the reporter also incorporated human C9orf72 intronic DNA immediately 5' of the hexanucleotide repeats. The reporter construct was integrated into the genome of HEK293t cells. We assessed the effect of depleting SUPT4H1 by RNAi interference on the transcriptome as a whole and on the DARP-EGFP reporter. Overall design: HEK293 cells stably expressing the intron-DARP-EGFP reporter were transfected with pools of siRNAs targeting SUPT4H1 or with scrambled control siRNAs. Starting from the same culture, six independent transfections were performed for each treatment side by side. A 3'-end tag library was generated using the Quant-seq (Lexigen) 3'-polyA library preparation kit and analyzed by Illumina sequencing.