title: Human iPSC-derived cardiomyocytes under hypoxia/normoxia, treated with bone marrow or cardiac stromal cell extracellular vesicles
design: Human iPSC-derived cardiomyocytes under hypoxia/normoxia, treated with bone marrow or cardiac stromal cell extracellular vesicles
library_construction_protocol: RNA-seq was performed on cultured human induced pluripotent cell derived cardiomyocytes (iPSC-CMs).  There are four groups, with three samples per group: H1,H2,H3. Negative control. Healthy, normoxic iPSC-CMs D1,D2,D3. Positive control. iPSCs cultured under hypoxic (0-1% O2) for 48h with 2% v/v PBS as vehicle control EV1,EV2,EV3. Treatment 1. iPSCs cultured under hypoxic (0-1% O2) for 48h, with cardiac stromal cell derived extracellular vesicles, provided at a dose of 67ng/µl (2% v/v) EV21, EV23, EV24. Treatment 2 iPSCs cultured under hypoxic (0-1% O2) for 48h, with bone marrow mesenchymal stromal cell (BM-MSC) derived extracellular vesicles, provided at a dose of 67ng/µl (2% v/v)  All EVs were isolated from cultured human cells using sequential centrifugation methods.  Cells were cultured using commercial EV-depleted FBS to avoid contamination of bovine EVs.   EVs were validated for CD81, CD9, ALIX positivity, and visualised by cryoEM.  Particle to protein ratios were not different between cardiac and bone marrow EV isolates.  Therefore EV doses were standardised so that the same dose by protein (67ng/µl) and EV number (~2,000 EVs per iPSC-CM) were added. RNA was extracted from hiPSC-CMs using a kit (Qiagen RNAeasy Micro, 217084) Agilent SureSelect XT HS2 mRNA Library Prep Kit