Multiple anti-tumor programs are activated by blocking BAD phosphorylation: RNA-seq of Bad+/+ or Bad3SA MMTV-PyMT breast tumors

Purpose: The goals of this study are to compare transcriptome (RNA-seq) differences between wild type and mutant Bad using MMTV-PyMT mouse breast cancer model. Methods: MMTV-PyMT breast cancer model tumors samples (6 independent mice for each genotype) were RNA-sequenced. Reads quality control and alignment to reference genome was done using fastp and STAR aligner respectively. Differentially expressed genes (DEGs) were computed using DESeq2. Overall design: Breast tumors for six independent mice for each genotype (PyMT-Bad+/+ and PyMT-Bad3SA end-point age matched mice), were harvested and snap-frozen in RNAlater® solution (Sigma Aldrich cat#R0901). To extract RNA, samples were thawed on ice and tissue retrieved with sterile forceps. Excess RNAlater® Solution was blotted away with an absorbent lab wipe. Tissue was then promptly lysed with a hand homogenizer (VWR Cat# 47747-370) and RNA extraction was carried out following the manufacturers protocol (QIAGEN's RNAeasy plus mini kit cat#74134). Elution was done using RNase free water. Nanodrop (NanoDrop™ 2000/2000c model) was used to measure the RNA samples concentration. 100ng/µl RNA samples were made by diluting 2000ng of RNA to a final volume of 20µl RNase free water. RNA samples were submitted to Génome Québec for sequencing using the Illumina NovaSeq 6000 sequencer in 100bp paired-end.