D6_SMI_2
Homo sapiens
Protocols: To separately isolate human ESC-derived cardiac lineage cells, including MCPs, intermediates, and differentiated cardiac cells, FACS sorting was performed with lineage-specific primary antibodies at the different time-points (day 3, 6, and 10) during cardiac differentiation. Cells were dissociated into single cells with Accutase for 5-10 min, washed in PBS, and blocked for 30 min in FACS buffer (1% bovine serum albumin and 10% horse serum in PBS) at 4°C. Staining for cell surface antigens was performed for 30 min at 4°C with the following primary antibodies: anti-platelet derived growth factor receptor-alpha (PDGFRA, a mesodermal marker; BD Biosciences), anti-PECAM1 (an endothelial cell marker; BD Biosciences), and anti-HCN4 (a pacemaker/conduction system cell marker; StressMarq). Cells were then fixed with 4% paraformaldehyde, permeabilized, blocked, and stained for intracellular antigens for 30 min at room temperature with the following primary antibodies: anti-ISL1 (a CPC marker; DSHB), anti-TNNT2 (a cardiomyocyte marker; Abcam), and anti-smooth muscle myosin heavy chain (SMMHC; a smooth muscle cell marker; Biomedical Technologies). After washing in PBS, cells were stained with Alexa-Fluor 405-, 488-, 546- or 647-conjugated secondary antibodies (BD Biosciences) specific to the appropriate species for 15 min at 4°C if the used primary antibody was not conjugated with a fluorochrome. Buffers were supplemented with 1:100 RNaseOUT Ribonuclease inhibitor (Thermo Fisher Scientific) if cells were to be recovered for RNA extraction. With a multicolor cell separation technique on a flow cytometer (FACSAria III) and FACS Diva software (Beckton Dickinson), single lineage populations, such as ISL1+PDGFRA+ MCPs (day 3), ISL1+Lin (TNNT2, HCN4, SMMHC, or CD31)+ intermediates (day 6) and ISL1-Lin+ differentiated cells (day 10) were separately sorted. FACS data were analyzed with FACS Diva and FlowJo software (Tree Star). Human ESCs (ES03; WiCell Research Institute) were maintained on feeder-free and 0.3 mg/ml Matrigel (BD Biosciences)-coated plates in mTeSR1 medium (STEMCELL Technologies) according to manufacturers' instructions. Cells were fed daily and passaged every 5-7 days with Dispase (STEMCELL Technologies). Media was supplemented with 5 μM ROCK inhibitor Y-27632 (Tocris) for 24 hours after splitting. For cardiac-directed differentiation, human ESCs maintained on Matrigel were dissociated into single cells with Accutase (STEMCELL Technologies) at 37 °C for 5-10 min and then seeded onto Matrigel-coated 12-well plates at 250,000-300,000 cells/well in mTeSR1 supplemented with 5 μM Y-27632 for 24 h (day −4). Cells were fed daily with mTeSR1 from day −3 to −1. At day 0, cells were treated with 12 μM GSK-3beta inhibitor CHIR99021 (Selleckchem) in RPMI medium supplemented with B27 minus insulin (RPMI/B27-ins; Thermo Fisher Scientific) for 24 h. At day 1, the GSK-3beta inhibitor was removed by replacing the media with fresh RPMI/B27-ins. At day 3, half of the medium in each well was changed to RPMI/B27-ins supplemented with 5 μM Wnt inhibitor IWP-2 (Tocris), all of which was replaced with fresh RPMI/B27-ins at day 5. At day 7, cells were switched to RPMI medium with B27 supplement (RPMI/B27, Thermo Fisher Scientific). Medium was replaced thereafter every 2 or 3 days. Immediately after FACS sorting, total RNA of the cells were extracted from the cell pellets using the RecoverAll Total Nucleic Acid Isolation Kit (Ambion), starting at the protease digestion stage of the manufacturer-recommended protocol. As previously reported (Hrvatin et al. 2014), the following modification to the isolation procedure was made: cells were incubated in digestion buffer for 1 hour at 50°C. Cell lysates were frozen at -80°C overnight before continuing the isolation according to the manufacturer's instructions. RNA was extracted according to the manufacturer's instructions. RNA integrity and concentration were measured using the Agilent RNA 6000 Nano kit with the 2100 Bioanalyzer instrument (Agilent). cDNA libraries of the human ESC-derived cardiac bulk populations were generated with the Smart-seq2 approach (Picelli et al., 2013; Ramsköld et al., 2012). 100 pg total RNA from bulk populations were mixed with 1 μl of anchored oligo-dT primer (10 μM, 5′-AAGCAGTGGTATCAACGCAGAGTACT30VN-3′, where “N” is any base and “V” is either “A”, “C” or “G”; Biomers.net) and 1 μl of dNTP mix (10 mM, New England Biolabs), denatured at 72 °C for 3 min and immediately placed on ice afterwards. After adding 5.7 μl of the first-strand reaction mix, containing 0.5 μl SuperScript II reverse transcriptase (200 U/μl, Invitrogen) and 0.1 μl template-switching oligo (TSO; 100 μM, Exiqon), reverse transcription reaction was carried out by incubating at 42 °C for 90 min, followed by 10 cycles of (50 °C for 2 min, 42 °C for 2 min). Then, the PCR master mix, containing 25 μl KAPA HiFi HotStart ReadyMix (2×, KAPA Biosystems) and 1 μl ISPCR primers (10 μM, 5′-AAGCAGTGGTATCAACGCAGAGT-3′; Biomers.net), was added to each sample tube, and pre-amplification PCR reaction was carried out as follows: 98 °C 3 min, then 15 cycles of (98 °C 15 s, 67 °C 20 s, 72 °C 6 min), with a final extension at 72 °C for 5 min. PCR products were purified using a 1:1 ratio of AMPure XP beads (Beckman Coulter). The library size distribution was checked on a High-Sensitivity DNA chip (Agilent Bioanalyzer). The expected average size should be around 1.5-2.0 kb, and we relied on the amount of cDNA comprised in the interval 300-9,000 bp plot. Five hundred picograms to 1 ng of cDNA were then used for the tagmentation reaction that was car¬ried out with 5 μl of tagment DNA enzyme of the Nextera DNA Sample Preparation kit (Illumina) at 55 °C for 5 min. To strip off the tagment enzyme, 5 μl of NT buffer (Nextera XT DNA sample preparation kit) was added to the 20-μl solution containing the tagmented DNA, and the mixture was incubated for 5 min at RT. Next, the whole volume was used for final enrichment PCR, along with 15 μl of Nextera PCR Primer Mix (NPM), 5 μl of Index 1 primers (N7xx), 5 μl of Index 2 primers (N5xx). A second amplification round was performed as follows: 72 °C 3 min, 95 °C 30 s, then 12 cycles of (95 °C 10 s, 55 °C 30 s, 72 °C 30 s), with a final extension at 72 °C for 5 min. Purification of PCR products was done with a 0.6:1 ratio of AMPure XP beads and samples. The quality of the final cDNA library was verified using a High-Sensitivity DNA chip and Bioanalyzer (Agilent) instrument. cDNA quantification was performed with Qubit High-Sensitivity DNA kit (Invitrogen). Libraries were diluted to a final concentration of 4 nM and pooled equally for sequencing.
ENA-FIRST-PUBLIC
2019-03-31T17:02:00Z
ENA-LAST-UPDATE
2019-01-28T16:26:13Z
External Id
SAMEA5278080
INSDC center name
KIS
INSDC first public
2019-03-31T17:02:00Z
INSDC last update
2019-01-28T16:26:13Z
INSDC status
public
Submitter Id
E-MTAB-7537:D6_SMI_2
broker name
ArrayExpress
cell line
ES03
cell type
mesodermal cell_smooth muscle intermediate (SMI)
common name
human
developmental stage
embryo
disease
normal
genotype
wild type phenotype
organism part
inner cell mass
phenotype
ISL1+SMMHC+ on day 6
progenitor cell type
embryonic stem cell
sample name
E-MTAB-7537:D6_SMI_2
scientific_name
Homo sapiens