WT_D0_rep1
Mus musculus
Protocols: Mouse embryonic stem cells at day 0 were detached from the plates and resuspended to a single cell suspension using Trypsin EDTA. For the depletion of MEFs (mouse embryonic fibroblasts), the cell suspension was transferred to 0.1% Gelatin-coated plates with an excessive volume of ESC medium and incubated for 20-30 minutes to allow MEFs to loosely attached to the plates, followed by carefully harvesting the ESC-containing supernatant. Cells were strained with a 40 µM cell strainer (Greiner #542040), washed three times and counted in Trypan blue using a Countess II Automated Cell Counter.  Embryoid bodies at D2 and D4 were harvested by spinning at 300 rpm for 5 min, washed twice with PBS and dissociated using StemPro Accutase (Invitrogen, #A1110501). After two washes using PBS supplemented with 10% of FBS, single cells were filtered through a 0.22 µM cell strainer and counted for downstream applications.   For D4 sorted samples, D4 dissociated EBs were incubated with 0.5 µg of Biotin anti-mouse CD309 (VEGFR2, Flk-1) antibody (Biolegend, #121904) and 1 µg of PE anti-mouse CD140a Antibody (Biolegend, #135905) for 1 X 106 cells in 100 µl of staining buffer (10% FBS in PBS). After an incubation of 30 minutes at 4°C and 3 washes with the staining buffer, 1 X 106 cells in 100 µl of staining buffer were incubated with the secondary antibody APC Streptavidin (Biolegend, 405207) for 15 minutes at 4°C, washed three times and resuspended in staining buffer. Cell sorting was performed using the BD FACSAria™ III Cell Sorter (BD Biosciences) and FACSAria™ II SORP.  Total RNA of WT and KO lines was extracted using ReliaPrep RNA Cell MiniPrep System (Promega, #Z6012) according to the manufacturer's instructions.  RNA-seq libraries were prepared according to Illumina TruSeq stranded mRNA protocol following manufacturer's instructions, starting with 200 ng of total RNA.
ENA first public
2026-01-15
INSDC center name
Erasmus University Medical Center
INSDC status
public
Submitter Id
E-MTAB-15595:WT_D0_rep1
broker name
ArrayExpress
cell line
ESC derived cell line
cell type
mouse embryonic stem cell
collection date
not collected
common name
house mouse
developmental stage
mouse embryonic stem cells (D0)
genotype
wild type genotype
geographic location (country and/or sea)
not collected
isolate
not applicable
sample name
E-MTAB-15595:WT_D0_rep1
scientific_name
Mus musculus
strain
129X1/SvJ