Illumina HiSeq 2500 sequencing; RNA-seq of Human, Sheep and Rat Mesenchymal Stromal Cells
RNA-seq of Human, Sheep and Rat Mesenchymal Stromal Cells
After incubation, cells were rinsed in PBS twice before being lysed directly in Buffer RLT Plus from the RNeasy plus micro kit (Qiagen, Victoria, Australia). Cells were cultured in a humidified incubator at 37ºC, with 5% carbon dioxide. Growth medium provided contains 1 g/L D-glucose (Gibco, Victoria, Australia) Dulbecco's Modified Eagle Medium (DMEM), L-Glutamine and 110 mg/L Sodium pyruvate, supplemented with 10% foetal bovine serum (Bovogen, Victoria, Australia) and 1% Penicillin-Streptomycin (Gibco, Victoria, Australia). Total RNA was extracted from lysed cells using the RNeasy Plus Micro kit (Qiagen, Victoria, Australia). Quality of RNA was determined on the 2100 BioAnalyzer (Agilent Technologies, Victoria, Australia). All samples of at least an RNA Integrity Number (RIN) of ≥ 8.0 and an A260/280 ratio of 1.8 – 2.0 were accepted. Library preparation was performed on 1 µg total RNA per sample with the Illumina TruSeq® Stranded mRNA kit according to manufacturer's protocol (Illumina, San Diego, California).