title: The transcriptome of human iPSC-derived cardiomyocytes
design: The transcriptome of human iPSC-derived cardiomyocytes
library_construction_protocol: All iPSCs were seeded on plates coated with Matrigel™ (Corning) and cultured in mTeSR™1 Medium (Stem Cell Technologies). Medium was changed daily. Cells were split every 3-4 days (when the colonies covered approximately 85% of the surface area of the culture dish) using PBS 0.5mM EDTA dissociation buffer. Before each differentiation process, iPSCs were thawed and maintained in culture for 2-3 passages. Before seeding, cells were incubated with ROCKi (Y-27632, 10μM, Stem Cell Technologies) for 1 h at 37ºC and then treated with Accutase (Sigma) for 7 min at 37ºC for dissociation. Microwell plates (AggreWell™800, Stem Cell Technologies) were used to generate aggregates. Cells were plated at a density of 0.9 x 106cells/well (3,000 cells/microwell) in 1.5 mL/well of mTeSR™1 supplemented with 10μM ROCKi. After 24 hours, the full volume of the medium was replaced and aggregates were maintained in mTeSR™1 without ROCKi for 48h. From this time point on (day 0 of differentiation), RPMI 1640 (Thermo Fisher Scientific) was used as the basal medium. During the first 7 days, cells were cultured in RPMI supplemented with 2%(v/v) B-27 minus insulin (Thermo Fisher Scientific). After day 7, cells were cultured in RPMI supplemented with 2%(v/v) B-27 (Thermo Fisher Scientific) until the end of differentiation. At day 0 of differentiation, the Wnt signaling pathway was activated using the GSK3 inhibitor CHIR99021 (Stemgent) at a final concentration of 11 µM. After 24 hours (day 1), full-volume medium replacement with RPMI + B27 minus insulin was performed. On day 3, half of the medium in each well was replaced and cells were supplemented with Wnt inhibitor IWP-4 (Stemgent) at a final concentration of 5 µM for two days. On day 5, the total volume of medium was changed. On day 7, the aggregates were flushed from the AggreWell™800 plate with RPMI + B27 and transferred to ultra-low attachment 6-well plates (Costar, Corning). On day 10, the full volume of medium was replaced. On day 13, aggregates were dissociated using 0,25% Trypsin-EDTA (Gibco) for 7 min at 37ºC. After dissociation, cells were washed with 2% fetal bovine serum (FBS) and 2mM EDTA in phosphate buffered saline (PBS, 0.1M), and incubated with VCAM1 antibody (BioLegend, 1:50) diluted in PBS/2% FBS for 15 min at 4ºC. Then, cells were washed and suspended in PBS/2% FBS for FACS sorting (BD FACSAria™ III Cell Sorter) using a 100μm nozzle at 4ºC. Purified VCAM1 positive cells were plated on wells coated with Matrigel, at a seeding density between 20,000 – 40,000 cells/cm2. For the remaining time in culture, two-thirds of the total volume of RPMI + B27 was replaced every 2–3 days.  RNA was extracted using NZYol (NZYTech®) and digested with DNase I (Roche®). At least 1µg of total RNA was obtained from each culture of iPSC-CMs at day 30 of differentiation.  After purification with poly-T oligo-attached magnetic beads, libraries were prepared using the NEBNext® Ultra™ Directional RNA Library Prep Kit.