RNA-seq of MCF7 no treatment ctrl
RNA sequencing analysis3  104 MCF-7 cells were seeded in a T25 flask, cultured overnight and 298 treated with 0.5 M NU-1 or DMSO for 48 h. Media was removed, cells were washed 2 times with PBS, followed by chemical disassociation with TrypLe (Thermo Fisher). Media was added to quench the TrypLe, the cell suspension was centrifuged at 300  g for 5 min and pellets were shipped to Applied Biological Materials for RNA isolation and total RNA sequencing. RNA was isolated using Trizol (Thermo Fisher). The quality of the RNA extraction was 304 assessed by gel electrophoresis. All the samples passed internal QC for library preparation. Sequencing libraries were prepared using Illumina TruSeq Stranded mRNA Library Preparation kit following manufacturers recommendations. The quality of the libraries was assessed using Qubit DNA assay, Agilent Bioanalyzer 308 and qPCR. All libraries passed internal QC. Sequencing was performed with an Illumina NextSeq system. To carry out the transcriptomic study, paired-end reads were aligned with the hg38 human reference genome by Hisat2. The abundance of genes was quantified by Htseq-count. Analysis of differentially expressed genes (DEGs) between NU-1 treated and control cells was performed with EdgeR in R studio, with dispersion value estimated as 0.01. Genes were considered significantly upregulated if they displayed fold change > 1.5 and P value < 0.1 and significantly downregulated if fold change < -1.5 and P value < 0.1, comparing 317 NU-1 treated and control cells. The DEG lists were subjected to GO analysis by Reactome and DAVID to detect enriched pathways.