mRNA expression profiling of human mesenchymal stromal cell samples grown in vitro under different conditions (foetal calf serum (FCS) based medium versus human platelet lysate (PL) based medium)
mRNA expression profiling of human mesenchymal stromal cell samples grown in vitro under different conditions (foetal calf serum (FCS) based medium versus human platelet lysate (PL) based medium)
Sternal bone marrow aspirates were obtained from healthy donors, following the informed consent. RNA was isolated using the miRNeasy Micro Kit (Qiagen) following the manufacturer instructions. Libraries for mRNA sequencing were prepared using the TruSeq stranded mRNA sample prep kit (Illumina). The starting material (100 ng) of total RNA was mRNA enriched using the oligodT bead system (Illumina). The isolated mRNA was subsequently fragmented using enzymatic fragmentation. Then first strand synthesis and second strand synthesis were performed and the double stranded cDNA was adenylated and Illumina sequencing adaptors ligated onto the fragments ends, and the library was purified (Agencourt AMPure XP). The polyA+ RNA stranded libraries were pre-amplified with PCR and purified (Agencourt AMPure XP). The libraries size distribution was validated and quality inspected on the 2100 Bioanalyzer (high sensitivity DNA chip, Agilent). High quality libraries were quantified using the Qubit Fluorometer (Life Technologies), the concentration normalized and the samples pooled according to the project specification (number of reads).