Illumina HiSeq 4000 paired end sequencing; mRNA-based generation of marmoset PGCLCs capable of differentiation into gonocyte-like cells
mRNA-based generation of marmoset PGCLCs capable of differentiation into gonocyte-like cells
iPS cells were dissociated using Accumax. PGCLCs, ovaries (E74, E82, and newborn), and testes (E87 and 22 dpp) were dissociated using 0.25% Trypsin EDTA. Adult testes (3yr10mnth) were digested by a stepwise treatment of collagenease1 (1mg/ml) and 0.25% Trypsin EDTA.  Marmoset iPSCs were cultured in the MEF-conditioned Primate ES cell medium (Reprocell, RCHEMD001) containing bFGF (Reprocell, RCHEOT003) and 1×Antibiotic-Antimycotic (Nacalai, 0289-54)8. iPSCs were dissociated into single cells for passaging by treatment with Accumax (Nacalai, 17087-54) at 37℃. We typically plated 5.0 × 104 cells into one well of 6-well cell culture plates in a conditioned medium containing iMatrix (FUJIFILM Wako, 892021) and 10 µM Y-27632 (Nacalai, 08945-42). iPSCs (lines 971 and 972) were induced from ear fibroblast cells. For the generation of PGCLCs from iPSCs, a lipofection-based mRNA transfection was used. The day before transfection (Day 0), 5.0 × 104 marmoset iPSCs were plated onto 12-well cell culture plates. The next day (Day 1), the medium was replaced with 500 µl of new conditioned medium. For transfection, two tubes containing 62.5 ul of OPTI-MEM (Invitrogen) were prepared. In one tube, RNAs [ 0.2 µg of SOX17 mRNA, 0.03 µg of P53DD mRNA, 0.75 µg of the mixture of E3, K3, B18R mRNAs (Reprocell)] were added. In the other tube, 1.5 µl of RNAiMax was added. After mixing well each tube, solutions in two tubes are mixed together. The mixture was left for 10 minutes. The mixture was mildly dropped onto one well of 12-well plate. The next day (Day 2), the medium was replaced with 500 µl of new conditioned medium, and RNA transfection was performed again. Medium change and RNA transfection were usually performed at 10 am and 4 pm, respectively.  On Day 3 morning (~10 am), the cells were dissociated using Accumax. ~5,000 cells were plated  onto each well of Nunclon™ Sphera™ 96-Well plate (Thermo Fisher Scientific, 174929) in 100 µl of aRB27 (1%B27/2 mM L-glutamine/1×Non-essential amino acids/1×Antibiotic-Antimycotic in Advanced RPMI medium) containing 50 mM EGF (R＆D systems, 236-EG-200), 100 mM SCF (R＆D systems, 455-MC-050), 10 ng/ml LIF (Merck, LIF1010), 400 mM BMP4 (R＆D systems, 314-BP-500), and 10 µM Y-27632. The medium change was performed every four days. For iPS cells, PGCLCs, and E82 ovary, the libraries were made using Chromium Next GEM Single Cell 3' GEM Library & Gel Bead Kit v3 (10x Genomics). Other libraries were made using Chromium Single Cell 3' Library and Gel Bead Kit v2 (10x Genomics). For iPS cells, PGCLCs, and E82 ovary, the libraries were made using Chromium Next GEM Single Cell 3' GEM Library & Gel Bead Kit v3 (10x Genomics). Other libraries were made using Chromium Single Cell 3' Library and Gel Bead Kit v2 (10x Genomics).