Ion Torrent Sequencing and DEG Analysis

Template preparation and chip loading was carried out using Ion 520™ and Ion 530™ Kit-Chef (Ion Torrent) on Ion Chef^TM Instrument (Ion Torrent). Sequencing was performed using Ion 520^TM Chip Kit (Ion Torrent) on Ion S5^TM System (Ion Torrent). Primary analysis of the sequencing data was performed using ampliSeqRNA plugin in the Torrent Suite™ Software v.5.10.1, as was published before.^26–28 Further analysis was performed on the EdgeR package from the open-source Bioconductor project. A matrix of gene-wise read counts was used as input. Normalization by trimmed mean of M values (TMM) was performed to adjust for different RNA compositions between libraries and library size. Genes from MCF-7 and MDA-MB-468 cells with less than 21.7 and 36.6 counts per million (CPM) (in any treatment or control group) were excluded from further analysis, respectively. This step was followed by differentially expressed gene (DEG) analysis under the GLM framework. Adjusted P-values for multiple testing, using Benjamini-Hochberg to estimate the false discovery rate (FDR), were calculated for the final estimation of DEG significance.²⁶ Genes with a fold change ≥1.5 or ≤0.67 and an adjusted P<0.05 were considered to be differentially expressed.

Raw sequencing data has been deposited in publicly available Sequence Read Archive (SRA) repository with links to BioProject accession number PRJNA675979 in the NCBI BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/).