RNA Extraction and Sequencing of Differentiated MSCs

Total RNAs was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and the quality of RNAs were evaluated by modified formaldehyde agarose gel electrophoresis and OD₂₆₀/OD₂₈₀ absorbance ratio detection using a Genova Nano spectrophotometer (Bibby Scientific; Cole-Palmer, Stone, UK). RNA-seq data from the osteoblastic or adipocytic differentiation induced MSCs were obtained at 7, 14 or 21 days using SMARTer Universal Low Input RNA kit for sequencing (Clontech Laboratories, Inc., Mountainview, CA, USA), according to the manufacturer's protocol. The Fast-QC software (Babraham Bioinformatics, Cambridge, UK; http://www.bioinformatics.babraham.ac.uk/projects/fastqc) was used to assess the data, including the quality value distribution of bases. RNA sequences were subsequently mapped to human genome sequences using TopHat software 1.3 release (Center for Computational Biology, John Hopkins University, Baltimore, MD, USA; https://ccb.jhu.edu/software/tophat/index.shtml) (16 (link)). Expression values of mRNAs were calculated based on the fragments per kilobase of exon, per million of fragments mapped and fragment length.