title: RNAseq of effects of high glucose environment on differentiated primary osteoblasts
design: RNAseq of effects of high glucose environment on differentiated primary osteoblasts
library_construction_protocol: Rats were euthanized with CO2 and femurs and tibias were dissected, cleaned of soft tissues, and the bone marrow (BM) was evacuated by centrifugation. Bone marrow stromal cells were enriched by plastic adherence for 2 days and expanded for additional 5 days for a total of 7 days before collecting, seeding, and using them in the experiments. The expansion was done in basal medium containing α-Minimum Essential Medium (αMEM (5.5 mM glucose), Gibco, Thermo Fisher Scientific, USA,) with 15% inactivated fetal bovine serum (iFBS, Gibco), HEPES (10 mM, Gibco), GlutaMAX™ (2 mM, Gibco), and Penicillin-Streptomycin (100 U/ml, 100 µg/ml, Gibco). During the expansion, the media was additionally supplemented with Amphotericin B (2.5 µg/ml, Gibco) and dexamethasone (10-8 M, Sigma, USA). All cell cultures were performed at 37°C and 5% CO2.  Bone marrow stromal cells were differentiated to osteoblasts for 10 days using osteogenic medium containing basal medium with 10% iFBS, supplemented with Na-β-glycerophosphate (10 mM, Fluka BioChemika, Switzerland) and L-ascorbic acid 2-phosphate (70 µg/mL Sigma, USA). BMSCs were seeded on 6-well plates at 40 000 cells per well in six replicates. Undifferentiated BMSCs were cultured for 24h at 300 000 cell per well (n=6) without the osteogenic supplementation. All cell cultures were performed at 37°C and 5% CO2.   High glucose was introduced by adding D-glucose to culture medium for up to 10 days. HG medium was made by adding 19.5 mM D-glucose (Sigma, USA) to the respective normoglycemic (NG) medium containing 5.5 mM glucose, resulting in the final glucose concentration of 25 mM.  For short-term exposure, HG was introduced to osteoblasts on day 9 of differentiation, one day before sample collection; or on day 7 of differentiation, three days prior to sample collection. For long-term exposure, HG was introduced on day 0 for the entire duration of differentiation (10 days). Culture medium was changed completely on days 4 and 7, and additionally on day 9 for the short-term exposure. Osteoblasts grown in NG media served as controls. Cell lysates were collected and RNA was isolated using Macherey-Nagel Nucleospin RNA Plus kit according to manufacturer's instructions with the following modifications. The samples were collected into the lysis buffer using a cell scraper and stored at -85°C until isolation. RNA lysates collected at day 10 were additionally homogenized with a homogenizer (UltraTurrax T25, Janke and Kunkel IKA-Labortechnik, Germany) in three 1-3 second bursts at 20 000 rpm prior to RNA isolation to ensure the breakdown of ECM. RNA was eluted in RNase-free water and stored at -85C. mRNA library preparation, RNA-seq, and bioinformatic analysis was purchased from Novogene, Ltd. (Cambridge, UK). In brief, RNA quality and integrity was first evaluated with BioAnalyser Agilent 2300 system. mRNA library suitable for RNA-seq was prepared via poly-A enrichment.