Illumina HiSeq 4000 sequencing; TFAP2A is required for efficient Primordial Germ Cell-like Cell induction
TFAP2A is required for efficient Primordial Germ Cell-like Cell induction
Embryoid bodies were dissociated using TrypLE, neutralised using MEF media and sorted to remove doublets and debries using a SONY SH800 sorter For the TFAP2A knockout and parental control line E-MTAB-11305 we used HUES9 iCRISPR and H9-TFAP2A lines ESC (https://www.pnas.org/doi/10.1073/pnas.1708341114#sec-3) which are derived from H9 cell line https://www.wicell.org/home/stem-cells/catalog-of-stem-cell-lines/wa09.cmsx. Embryoid bodies were aggregated from PreME cells and treated with BMP to induce PGCLC fate according to Kobayashi et al 2017. PreME/ME protocols were optimised based on previous conditions. For the basal medium, we used aRB27 medium which is composed of Advanced RPMI 1640 Medium (Thermo Fisher Scientific) supplemented with 1% B27 supplement (Thermo Fisher Scientific), 0.1 mM NEAA, 100 U/ml Penicillin- 0.1 mg/ml Streptomycin, 2 mM L-Glutamine. For PreME/ME induction, trypsinized Conv-hPSC were seeded on vitronectin-coated dish at 200,000 cells/well in 12 well plate and cultured in PreME/ME induction medium for 6-36 h as indicated in the figures.   PreME/ME induction medium was composed of aRB27 medium supplemental with 100 ng/ml Activin-A (SCI), 3 ŒºM GSK3i and 10 ŒºM of ROCKi.   Single Cell 3' 10X V3 Single Cell 3' 10X V3