Determining the transcriptional regulation of CDK4/6 inhibitor resistance in estrogen receptor-positive breast cancer

CDK4/6 inhibitor (CDK4/6i) resistance is a pressing clinical problem for patients with ER+ breast cancer. Previous work identified CDK6 overexpression as one critical determinant of acquired resistance to CDK4/6 inhibition. This dataset interrogates CDK6 interactors/recruiters to determine their role in transcriptional regulation of CDK4/6i resistance mechanisms and will provide novel therapeutic strategies to overcome resistance. Overall design: RNA was extracted from cultured cells treated with vehicle (Palcociclib-Resitant 100nM) or the addition of JQ1 (300nM - 24hr) using the RNeasy Mini Kit (Qiagen). Prior to RNA sequencing, RNA was treated with DNase using RNase-Free DNase Set (Qiagen). rRNA depletion was performed from 100ng of purified RNA using QIAseq FastSelect rRNA HMR reagents according to manufacturer's protocol. Libraries were prepared using Roche Kapa Biosystems RNA HyperPrep sample preparation reagents on a Beckman Coulter Biomek i7. Finished dsDNA libraries were quantified by Qubit fluorometer and Agilent TapeStation 4200. Uniquely dual indexed libraries were pooled in an equimolar ratio and shallowly sequenced on an Illumina MiSeq to further evaluate library quality and pool balance. The final pool was sequenced with paired-end 150bp reads on an Illumina NovaSeq 6000at the Dana-Farber Cancer Institute Molecular Biology Core Facilities. Sequenced reads were aligned to the UCSC hg38 reference genome assembly and gene counts were quantified using STAR (v2.7.3a).