MCF-7 breast cancer cells treated with ZnSO4 for 120 minutes replicate 3
MCF-7 cells (human breast adenocarcinoma, ATCC Cat. No. HTB-22) were cultured in DMEM (Life Technologies, Australia) supplemented with 10% foetal bovine serum (FBS) (Life Technologies, Australia), 1% antibiotics penicillin/streptomycin and L-glutamine (Life Technologies, Australia). Cells were incubated at 37 C with 5% CO2 and observed daily until 80% confluence. The cells were then washed with phosphate-buffered saline (PBS), trypsinised, counted using a haemocytometer (LW Scientific) and seeded into appropriate flasks for experimentation. MCF-7 cells were grown in 25 cm2 flasks for 32 h at 80% confluency, and then the medium was aspirated and replaced with 5.9 mL of fresh complete medium. ZnSO4 at 60x stock concentration was prepared and filter-sterilised through a 0.22 m filter. Each flask was treated with either 100 L of the predetermined IC50 dose of ZnSO4 (320 M), or 100 L of H2O to serve as a control. Flasks were incubated at 37 C with 5% CO2 for T0, T30, and T120 min respectively for each treatment. Immediately after the incubation, medium was discarded, and 1 mL of TRIzolTM reagent (Thermo Fisher Scientific) added directly into the flask. After mixing several times, the lysate was homogenised and transferred to 2 mL microfuge tubes. The resultant lysate was incubated for 5 min at room temperature to allow complete dissociation of the nucleoproteins complex from the nucleic acids. Then, 200 L of chloroform (Sigma-Aldrich, Australia) was added, followed by a further 3 min incubation. Samples were centrifuged for 15 min at 12,000 g, 4 C to separate into three phases. The experiments are carried out in triplicates. The RNA containing the upper phase was transferred to a fresh microfuge tube and precipitated by adding 0.5 mL of isopropanol (Sigma-Aldrich, Australia). After 10 min incubation, the sample was centrifuged for 10 min at 12,000 g, 4 oC. The supernatant was then discarded, and the RNA pellet was washed in 1 mL 75% ethanol (Sigma-Aldrich, Australia) by vortexing for 5 sec then centrifuged for 5 min at 7,500 g, 4 oC. The resultant RNA pellet was allowed to air-dry for approximately 20 min, then the RNA pellets for each treatment were re-constituted with RNase-free water (Sigma-Aldrich, Australia), and quantified by spectrophotometer (NanoDropTM 2000). RNA samples with the concentration at 100 ng/L were prepared and sent to the Ramaciotti Centre for Genomics (University of New South Wales, Australia) for next-generation RNA sequencing. The quality of the RNA samples was further checked at Ramaciotti Centre with the Tapestation (Agilent) for integrity and the Nanodrop for quantification and purity. Samples were then prepared using the TruSeq Stranded mRNA-seq kit (Illumina) using 1 g of total RNA as input. The number of PCR cycles was reduced from 15 to 12, to minimise PCR duplicates in the data. The generated libraries had a median insert size of 180bp (range 100-370bp) and were pooled in equal amounts for the sequencing run. One sequencing run with NextSeq 500 was performed using a NextSeq 500/550 High Output v2 kit and sequenced to produce 75bp single reads. Fastq files were generated in Basespace using bcltofastq 2.18. The run generated over 450M reads (40-50M per sample), and quality specification Q30 was 93%. Bioinformatic analysis to identify differentially expressed genes was performed at the Systems Biology Initiative (UNSW). The RNA reads were mapped to the Ensembl Homo sapiens genome (GRCh38). The mapping was performed with Tophat2 (v 2.0.12) and Bowtie2 (v 2.2.3). The featureCounts function of Subread (v 1.4.6-p5) was used to generate counts of reads uniquely mapped to annotated genes using the GRCh38 gtf file. Differential expression analysis was performed using the Bioconductor packages edgeR (v 3.18.1). Generalised linear models were used for differential expression analysis. In all cases, differentially expressed genes were defined as those genes with a Benjamini-Hochberg corrected p-value less than 0.05. The fold change (FC) is expressed in Log2FC. The genes with over 0.6 of Log2FC are identified as the up-regulated genes, whilst the genes with lower than -0.6 of Log2FC as the down-regulated genes.