Bulk RNA seq timecourse of KOLF2.2J hiPSC after Auxin-induced protein degradation of specific gene products

This study investigates the impact of 2 gene products (RBBP4, CHD1) on transcriptional programming in growing hiPSC at 0, 6 & 24 hours after acute protein depletion. Crispr editing was used to add an auxin-dependent degron to the C-termini of gene products expressed from the endogeneous loci and to insert an auxin-responsive degradation E3 ligase to the AAVS locus. Rapid (<30 min), specific target protein degradation in KOLF2.2J hiPSCs was induced by a synthetic auxin derivative (5-Ad-IAA) and bulk RNA-seq was performed at timepoints to define early (6 hr) and later (24 hr) downstream transcriptional dependencies. Crispr editing was used to introduce an auxin dependent E3 ligase (atOsTir1) into the AAVS locus of the parental KOLF2.2J hiPSC line. Cells were subsequently Crispr-edited to create independent clones with an auxin-specific degron added to the C-terminus of individual target gene products expressed from endogenous loci. Rapid degradation of target proteins was induced by treatment with the synthetic auxin derivative, 5-Ad-IAA, and protein loss was confirmed by Western blotting. Control cells were treated with DMSO. Bulk RNA-seq was performed in duplicate at 0 hr, 6hr, and 24 hr to evaluate transcriptional responses.