GSM4654762: hPGCLCs-1; Homo sapiens; RNA-Seq
hiPSCs and iMeLCs were harvested. The d5 floating aggregates containing hPGCLCs were incubated with 0.1% Trypsin-EDTA (Gibco) in PBS for 15 min at 37 °C with periodical pipetting. After quenching the reaction by addition of FBS and pipetting, cells were strained through a 70 µm nylon cell strainer (Thermo Fisher Scientific). The AG+ cells were sorted by FACSAria Fusion (BD Biosciences) and collected in an Eppendorf tube. xrTestes were treated with 0.1% Trypsin-EDTA in PBS for 15 min at 37 °C with periodical pipetting.  After quenching the reaction by addition of FBS and pipetting, cell suspension was washed with 0.1% BSA fraction V in PBS and strained through a 70 µm nylon cell strainer. AG+/-VT+/-PC+/- cells were sorted by FACSAria Fusion and collected in CELLOTION (Amsbio). Fetal gonads were cut into small pieces (~1mm2), then digested with collagenase type IV for 8 min at 37 ºC. Tissues were centrifuged at 200 g for 5 min and washed with Hank's Balanced Salt Solution (Thermo Fisher Scientific). Tissues were then digested with 0.25% trypsin/EDTA and DNase I for 8 min at 37 ºC. After the digestion was quenched by adding 10% of fetal bovine serum, cells were strained through nylon cell strainer (70μm in pore size). The cells were then pelleted by centrifugation at 200 g for 5 min, which were resuspended in the MACS buffer (PBS containing 0.5% BSA and 2 mM EDTA). Dead cells were subsequently removed by Dead cell removal microbeads (Miltenyi Biotec). Flow-through cells were pelleted by centrifugation at 600 g for 15 min and resuspended in PBS containing 0.1% BSA Cells were loaded into Chromium microfluidic chips with Chromium Single cell 3ʼ v3 chemistry and used to generate single cell gelbead emulsions (GEMs) using Chromium controller (10x Genomics) according to manufacturer's protocol. All subsequent cDNA amplification and library construction steps were performed according to manufacturer protocol.