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. 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.