nuclear RNA-seq of MCF10A cells: siAPOBEC3A pool siRNA treatment biol rep 2
MCF10A cells (ATCC, CRL-10317) were subcultured in Dulbeccos modified Eagles medium/nutrient mixture F-12 (Gibco, 1130-032) containing horse serum (Gibco, 16050), 10 g/mL insulin (Sigma, I1882), 0.5 g/mL hydrocortisone (Sigma H0135), 100 ng/mL cholera toxin (Sigma, C8052), and 20 ng/mL epidermal growth factor (Peprotech, AF-100-15). Cells were maintained at 37C, in a humidified atmosphere with 5% CO2. At least 3 million cells were seeded for each treatment on Day 0. 33nM siRNAs (Dharmacon/Horizon Discovery) were added on Day 1 per manufacturer's protocol. Cells continued to grow for 72 more hours. Nuclei were purified after swelling cells and then using a 7mL dounce. RNA from nuclei was harvested using TRIzol reagent per manufacturer's protocol. 1 microgram of RNA was given to the Yale Center for Genome Analysis for library prep. Illumina RNA-seq library prep was performed without polyA enrichment or ribosomal RNA depletion steps.