Prdm1

The in situ PLA was performed on fixed MO-DCs with Duolink in situ Detection Reagents Red (Sigma Aldrich) according to the manufacturer's instructions. Briefly, cells were fixed with 4% paraformaldehyde (PFA) at room temperature and washed with PBS. Samples were permeabilized with 0.5% Triton-X-100 in PBS and blocked by blocking solution (provided by the kit) for 1 h at 37°C. Primary antibodies against NonO (Cat# sc-376865, Santa Cruz), hnRNPM, TP53BP1, PRDM1, V5, Flag or normal rabbit IgG (Cat# 2729, Cell Signaling Technology) were incubated overnight at 4°C. The samples were washed twice for 5 min with buffer A (provided by the kit), followed by incubation with the PLA probes (Sigma Aldrich) for 60 min at 37°C. Subsequent ligation for 30 min at 37°C and amplification for 100 min at 37°C were performed. Finally, the samples were mounted using Duolink in situ Mounting Medium with DAPI (Sigma Aldrich). Z-Stacks Images were captured using a 60X oil objective on Zeiss Apotome 2 microscope and LSM 880 confocal microscopy (Carl Zeiss Microscopy). Three-dimensional foci counting analysis was performed with Imaris software (Imaris v8.0.2).

Following induction of EpiLCs and PGCLCs as previously described, after two days of incubation with PGCLC medium, cells were stained with SSEA1 and CD61 for 30 min and then washed with MACS buffer (1% BSA, 1 mM EDTA in PBS). Cells were collected by centrifugation at 400 g for 5 min at 4 °C and fixed with 4% PFA for 10 min. The cells were then washed with PBS and permeabilized with cold 0.1% Triton-X-100 in PBS (PBST) on ice for 10 min. After washing with PBST, cells were blocked with 2% BSA in PBST at room temperature for 30 min. Cells were then washed with PBST and resuspended in MACS buffer with the primary antibody (Prdm1, Cell Signaling, 9115S) for 1.5 hours at 4 °C in the dark. The cells were then washed with PBST and stained with secondary antibody for 30 min at 4 °C. Next, after washing with PBST and then MACS buffer, the cells were resuspended with MACS buffer, and large clumps of cells were removed using a cell strainer (Falcon^™ 352235). Finally, cells were analyzed on flow cytometers.