2024-06-01
ENA first public
ERC000011
ENA-CHECKLIST
Instituto de Medicina Molecular João Lobo Antunes
INSDC center name
public
INSDC status
ERS18324102
SRA accession
E-MTAB-13850:Sample 1
Submitter Id
ArrayExpress
broker name
iPSC derived cell line
cell_line
cardiac muscle cell
cell_type
not collected
collection date
human
common name
Protocols: ll 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. At day 30, differentiated cells were dissociated using 0.25% Trypsin-EDTA (Gibco) for 5 min at 37ºC. After stopping the reaction, dissociated cells were resuspended in PBS/0.1% bovine serum albumin (BSA; Life Technologies), filtered through a 100 μm pore filter and stored in ice.  Viable cell suspensions (1x106 cells/mL) were partitioned into Gel Beads-in-emulsion (GEMs), in order to capture approximately 10000 cells in single cell droplets, using the Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit (version 3, 10X Genomics, PN-1000092) and Chromium Single Cell B Chip Kit (10X Genomics, PN-1000074). Barcoded cDNA was extracted from the GEMs by Post-GEM RT-cleanup, PCR amplified and subjected to quality control and quantification.  Single Cell 3ʹ Gene Expression libraries from single cells was then performed using Chromium i7 Multiplex Kit (PN-120262). All the steps were performed according to the manufacturer’s protocol.
description
day 30 of differentiation
developmental stage
wild type genotype
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
Homo sapiens
scientific_name
male
sex
Sample 1
title