Transcriptomic and miRNA analysis of control versus treated HCT-116 cells with vitamins A and D

mRNA-seq: HCT-116 colon cancer cells were grown and maintained and treated with a combination of Vitamin D2 and D3 and all trans-retinoic acid (ATRA) at 1 µM 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. miRNA-seq: HCT-116 cells were cultured in McCoy’s 5a Medium (Gibco, Life Technology, Grand Island, NY, USA) supplemented with 10% FBS and 1% Penicillin/Streptomycin and incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air. At 80% confluence, cells were harvested by adding 0.25% trypsin/EDTA and counted by means of Trypan blue and hemocytometer. The cells were then re-suspended at appropriate concentration and plated in 96 or 6 well plates for cellular assays. Cell were treated with ATRA+D2+D3 at the IC50 concentration (1mM), then harvested and RNA isolated using Trizol Control samples in triplicate versus treated samples in triplicate