A traceless system for small molecule control of both transgene and endogenous gene expression

This project investigates an acyclovir-controlled poison exon system (Cyclone) for traceless gene regulation. We performed RNA-seq on mammalian cells treated with acyclovir or branaplam to compare their effects on global gene expression and splicing. These data support our findings that acyclovir has minimal impact on cellular physiology while enabling robust gene induction, in contrast to branaplam.HEK293T cells were plated in 10-cm tissue culture dishes (Corning 430167) and cultured under standard conditions. Four experimental conditions were established in biological triplicate: (1) DMSO control for acyclovir treatment, (2) DMSO control for branaplam treatment, (3) acyclovir treatment (250 uM), and (4) branaplam treatment (100 nM). After three days of treatment, total RNA was extracted using TRIzol LS Reagent (Invitrogen 10296010), treated with DNase I (NEB M0303S), and purified using the RNA Clean Concentrator-25 kit (Zymo R1018). RNA-seq libraries were prepared with the Illumina TruSeq Stranded Total RNA Library Preparation Kit (with rRNA depletion) at the Weill Cornell Medicine Genomics Resources Core Facility. Sequencing was performed on an Illumina NovaSeq X Plus platform, generating about 70 million 150-bp paired-end reads per sample.