Gmem medium

The RVFV-4s vaccine virus used in the presented studies is based on RVFV strain 35/74^{24 (link)} and referred to as vRVFV-4s. A master seed virus (MSV) of vRVFV-4s was prepared by amplification of the virus in BSR-T7 cells, grown in GMEM medium (Gibco), supplemented with 5% foetal calf serum (FCS, SAFC), 4% tryptose phosphate broth (Gibco), 1% non-essential amino acids (Gibco) and 0.01% Gentamicin. The MSV was passed 2–5 times in BSR-T7 cells and was subsequently used for efficacy studies with goats, calves and lambs (Table 1). Identity of the vRVFV-4s virus batches was confirmed by conventional RT-PCR and next-generation sequencing. For homologous challenge, recombinant RVFV strain 35/74 (rRVFV 35/74) was used and for heterologous challenge wild-type RVFV strain ZH501 was used. rRVFV 35/74 was propagated in BHK-21 cells, which were grown in CO₂-independent medium (Gibco), supplemented with 5% FCS (Gibco), 1% l-glutamine (Gibco) and 1% antibiotic/antimycotic (anti/anti, Gibco). RVFV strain ZH501 was propagated in Vero E6 cells that were grown in EMEM (Gibco), supplemented with 5% FCS (Gibco) and 1% anti/anti. For the virus neutralization assay, BHK-21 cells were used, which were grown in GMEM medium, supplemented with 5% FCS, 1% l-glutamine and 1% anti/anti.

Naive-hiPSCs colonies were separated as single cells with accutase and seeded in ultra-low attachment U-bottom 96 well plate (Corning, New York, USA) at a density of 8000–10,000 cells/well in PGC-LCs medium. Then naive-hiPSCs were centrifuged 120 g for 2 min to induce embryoid bodies (EBs) formation. Simultaneously of EBs formation, naive-hiPSCs differentiation into PGC-LCs was induced by culturing EBs in GMEM medium (thermofisher) supplemented with 15% KSR (thermofisher), 2 mM L-Glutamine (thermofisher), 0,1 mM nonessential amino acid (thermofisher), 1 mM sodium pyruvate (Sigma), 50 µM mercaptoethanol (thermofisher), 100 U/ml Penicillin—100 U/ml Streptomycin (Sigma) and cytokines 500 ng/ml BMP4 (TEBU-Bio), 1 µg/ml LIF (Peprotech), 100 ng/ml SCF (TEBU-Bio), 50 ng/ml EGF (TEBU-Bio) and 10 µM ROCK inhibitor (TEBU-Bio). After 4 days, EBs were observed in the center of each well. Three to five EBs were formaldehyde-fixed and paraffin-embedded for each clone studied, then paraffin sections of EBs were used for immunohistochemistry experiments.

All cell lines used in this study originate from E14TG2a.4 mESC (mouse Embryonic Stem Cells, Mus musculus strain 129/Ola, male) with integrated gene targeting vector pAW2 at the ROSA26 locus to enable recombinase-mediated cassette exchange (Zhou et al., 2013 (link)). mESC cells were grown in G-MEM medium (ThermoFisher) supplemented with 5% embryonic stem cell-qualified FBS (ThermoFisher), 5% Knockout Serum Replacement (ThermoFisher), 1x non-essential amino acids (ThermoFisher), 1x sodium pyruvate (ThermoFisher), 100 μM β-mercaptoethanol (Sigma-Aldrich) and 1000 U/mL LIF (Millipore). Cells were sub-cultured every two days into dishes coated with 0.1% gelatine and kept at 37°C in a 5% CO₂ water-saturated incubator.
For depletion of ARID1A, cells were seeded at ∼20,000 cells per cm² and 500 μM auxin analog 1-naphthaleneacetic acid (NAA, Sigma-Aldrich) in 1M sodium hydroxide was added for the indicated time before harvesting the cells after two days of growing.

Calu-3 cells (American Type Culture Collection (ATCC), Manassas, VA, USA), hpF, and MDCK cells (European Collection of Authenticated Cell Cultures (ECACC), UK Health Security Agency, Salisbury, UK) were cultivated in MEM medium containing GlutaMAX (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1 mM sodium pyruvate (Thermo Fisher Scientific), and 10% FBS (Bio&Sell, Feucht, Germany), in DMEM medium (high glucose) containing GlutaMAX and sodium pyruvate (Thermo Fisher Scientific) with 10% FBS, and GMEM medium (Thermo Fisher Scientific) with 1% tryptose No. 2 (NEOGEN, Lansing, MI, USA), and 10% FBS, respectively. Media were changed every two to three days. All cells were maintained under standard culture conditions in a humidified incubator containing 5% CO₂ at 37 °C.