design: Transcriptomic profiling reveals key early response genes during GDF6-mediated differentiation of human adipose-derived stem cells to nucleus pulposus cells
library_construction_protocol: All experiments and procedures carried out in this study were performed with relevant approval from the National Research Ethics Service and The University of Manchester. Adipose tissue was obtained from donors undergoing hip replacement surgery, with each donor providing full written informed consent. The adipose derived stem cells (ASC) extraction process was undertaken as previously described [PMID: 32489577]. ll experiments and procedures carried out in this study were performed with relevant approval from the National Research Ethics Service and The University of Manchester. Adipose tissue was obtained from donors undergoing hip replacement surgery, with each donor providing full written informed consent. The adipose derived stem cells (ASC) extraction process was undertaken as previously described [PMID: 32489577]. Donor ASCs used for experimentation were expanded in culture medium containing: 110mg/L sodium pyruvate, 1000 mg/L glucose and further supplemented with final concentrations of 10% (v/v) FBS, 2mM GlutaMAX (Life Technologies), 50μg/mL ascorbate, 100U/mL penicillin, 100μg/mL streptomycin and 0.25μg/mL amphotericin (subsequently termed standard culture medium). ASCs were grown in ambient incubator conditions (37°C, 5% CO2, 20% O2) until sufficient cell numbers were obtained. All donors were used between passages 3-5 and around 14 days in culture. ASCs were seeded in flasks or multiwell plates at a density of 7.5x103 cells/cm2 (70-80% confluence) and allowed to adhere for 24hrs. Cells were washed twice in PBS and serum starved in FBS-free standard culture medium (SFM) for a further 24hrs. On the day of GDF6 stimulation, medium was aspirated, cells washed twice in PBS and stimulated with 100ng/mL of GDF6 (Peprotech) in SFM as previously described [PMID: 32489577]. Samples were taken at 4 timepoints (0h, 2h, 6h, and 12h). Total RNA was extracted using TRIzol according to the manufacturers protocol, then resuspended in 20µL TE buffer.  Libraries were generated using the TruSeq® Stranded mRNA assay (Illumina, Inc.) according to the manufacturer's protocol