mRNA-seq study performed on MCF-7 breast cancer cells treated with a combination of vitamins A and D

MCF-7 breast cancer cells were grown and maintained and treated with a combination of Vitamin D2 and D3 and all trans-retinoic acid (ATRA) at 1.5 µg/ml for four hours. The cells were harvested, RNA was isolated and analyzed for integrity using Agilent 4200 TapeStation and RNA Screen Tape. RNA-seq libraries were prepared using a Universal Plus mRNASeq kit, and library fragment size distribution was confirmed by electrophoresis using a 2200 TapeStation system with D1000 ScreenTape. Sequencing was carried out on NovaSeq 6000, SP flowcell, 2x50 nt reads. General quality-control metrics were obtained using FastQC and raw reads were aligned to human reference genome hg38 using STAR and BWA MEM. ENSEMBL genes were quantified using FeatureCounts, and differential expression statistics were computed using edgeR. Specific gene expression was measured and verified using qPCR. Overall design: mRNA-seq study performed on MCF-7 breast cancer cells treated with a combination of vitamins A and D. Control samples in triplicate versus treated samples in triplicate.