RNA-seq of mouse wild-type and I830077J02Rik,C130074G19Rik,1500009L16Rik,D630003M21Rik,D430041D05Rik and A530016L24Rik gene knockouts in Flk-1+/Pdgfra+ and Flk-1+/Pdgfra- sorted populations at day 4 of embryoid body differentiation
RNA-seq of mouse wild-type and I830077J02Rik,C130074G19Rik,1500009L16Rik,D630003M21Rik,D430041D05Rik and A530016L24Rik gene knockouts in Flk-1+/Pdgfra+ and Flk-1+/Pdgfra- sorted populations at day 4 of embryoid body differentiation
Embryoid bodies at 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.  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.