smalRNA seq of IPSC-cardiomyocytes treated with exosomes before hypoxic stiumli
smalRNA seq of IPSC-cardiomyocytes treated with exosomes before hypoxic stiumli
Cells, in 96-well plate, were washed gently with PBS (1X) before QIAzol lysis reagent (100 uL) (Qiagen, #79306, Hilden, Germany) was added to the cells and the lysate was collected and pooled in to three samples for the control group and three samples for the EXO-preconditioned group   Ncyte™ Cardioplate™ Maestro™ MEA 96 with pre-seeded IPSC-cardiomyocytes (Ncardia, # Nc-K-NCP-M96, Leiden, NL) was maintained in in Cardiomyocyte Culture Media (Nc-M-CMCM-250, Ncardia, Leiden, NL) at 21% O2, 5% CO2 in a humidified atmosphere at 37C, until stable electrophysiological activity was observed using the Multi-electrode array (MEA) systems. exosome-precondition group (MEA_EXO_RNA) was administred exosomes 24 hpurs before 16 hours of hypoxic stimulus. MEA_CONTROL_RNA was not exposed to exosomes (only medium change) 24 hours before 16 hours of hypoxic stimulus.  RNA was isolated using the miRNeasy Micro Kit (Qiagen, #217084, Hilden, Germany) following the manufacturer's instructions. RNA sample quality control was performed using the RNA 6000 Pico Kit (Agilent, #5067-1513, Santa Clara, CA, USA) with a 2100 Bioanalyzer System (Agilent, #G2939BA, Santa Clara, CA, USA) following the manufacturer's instructions. Then, a NEXTFLEX® Small RNA-Seq Kit v4 with UDIs (PerkinElmer, #NOVA-5132-44 and #NOVA-5132-32, Waltham, Massachusetts, USA) was used to generate the sample library, and a 4200 TapeStation system (Agilent, Santa Clara, CA, USA) was used for quality control of the sample library. Based on the TapeStation data, sample concentrations were adjusted and then quantified using a Qubit Q32853 with the Qubit™ dsDNA BR Assay Kit (Invitrogen, #Q32850, Waltham, Massachusetts, USA).