title: Illumina HiSeq 4000 paired end sequencing; RNA-seq of mesenchymal stromal cells cultured in the presence of breast cancer cell secretomes
design: RNA-seq of mesenchymal stromal cells cultured in the presence of breast cancer cell secretomes
library_construction_protocol: To retrieve cells, hydrogels were degraded in 0.5 mg/mL collagenase A (Sigma, 11088793001) at 37 °C for 60 min.  Human bone marrow-derived mesenchymal stromal cells (MSCs) were isolated as described previously (https://www.ncbi.nlm.nih.gov/pubmed/25020062) from bone marrow aspirates of healthy donors (n = 4) obtained during orthopedic surgical procedures after informed consent and in accordance with the local ethical committee (University Hospital Basel; Prof. Dr. Kummer; approval date 26/03/2007 Ref Number 78/07). MSCs are primary, wild type cells. MSCs were maintained in MEM alpha (with nucleosides, Gibco) supplemented with Fetal Bovine Serum (FBS) (10%, Gibco), penicillin (100 U ml-1, Gibco), streptomycin (100 µg ml-1, Gibco), and FGF-2 (5 ng ml-1, PeproTech) at 37°C in a humidified atmosphere at 5% CO2. Mesenchymal stem cells were grown in PEG hydrogels for 7 days in the presence of 2% serum plus serum-free media or a breast cancer cell secretome. Total RNA was isolated from cells using the RNeasy Micro Kit following the manufacturer's instructions (Qiagen, 74004). We used NEBNext Ultra RNA Library Prep Kit with PolyA selection for library preparation. A detailed protocol can be found here https://www.neb.com/-/media/catalog/Datacards%20or%20Manuals/manualE7530.pdf.
attributes.tag: Experimental Factor: stimulus
attributes.value: MDA-MB-231 brain-tropic secretome