Chicken serum

Human WT (ATCC, CRL-4000), TP53^−/− (ref. ^{35 (link)}), TP53^−/−/BRCA1^−/− (ref. ^{35 (link)}), FEN1^−/−, XRCC1^−/− (ref. ^{53 (link)}) and XRCC1^−/−/APE1^−/− hTERT RPE-1 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM/F12, Merck) supplemented with 10% fetal calf serum (FCS). Human WT (ATCC, HTB-96) and FEN1^−/− U2OS cells, and Rnaseh2b^−/− mouse embryonic fibroblasts^{54 (link)} were cultured in DMEM (Gibco) with 10% FCS and 2 mM l-glutamine (Gibco). All above cells were grown under 3% oxygen. The generation of FEN1^−/− U2OS and RPE-1 cells is described below, and the generation of APE1^−/− cells will be described in detail elsewhere. Chicken WT and FEN1^−/− (ref. ^{29 (link)}) DT40 cell lines were cultured in RPMI 1640 medium supplemented with 10% FCS, 1% chicken serum (Gibco), 2 mM l-glutamine and 10 µM β-mercaptoethanol (Gibco). All growth media was supplemented with penicillin (100 units per ml)/streptomycin (100 mg ml⁻¹) (Merck) and all cells were grown at 37 °C.

The JM8.N4 cell line, derived from the mouse strain C57BL/6N [30 (link)], was cultured on 0.1% gelatin coated plates (Corning NY, USA) in Knockout DMEM (Gibco, UK) containing 2mM L-Glutamine (Gibco, UK), 0.05mM β-mercaptoethanol (Sigma Dorset, UK), 10% fetal bovine serum (Biosera East Sussex, UK) and 1000U/ml LIF (ESGRO, Millipore Watford, UK), at 37⁰ and 5% CO_2, changing medium daily. Cells are detached with 0.1% trypsin (Gibco, UK), containing 1% chicken serum (Gibco, UK), 0.6 mM EDTA (Sigma Dorset, UK) and 0.1% D-glucose (Sigma Dorset, UK) and passaged every two to three days. The E14 mESC line (129P2 background) was cultured in the same way but using GMEM with the addition of pyruvate and keeping the cells in 7% CO_2.

The MC29 bone marrow-transformed chicken macrophage-like cell line HD11 [42 (link)] was maintained in Roswell Park Memorial Institute (RPMI) 1640 medium added GlutaMAXTM-I, 25 mM HEPES (Gibco, Carlsbad, CA, USA), 2.5% chicken serum, 10% heat-inactivated foetal bovine serum (ThermoFischer, Gibco, South America) and 25μg/mL gentamicin. The HD11 cells were incubated at 37 °C in an atmosphere of 5% CO₂. Cell concentrations were adjusted, and aliquots of cells (4 × 10⁶ cells/mL) were kept on ice until use. Prior exposure to bacteria, the culture medium of each sample was replaced with RPMI without antibiotics. The HD11 cell suspension was seeded into each well at 100 μL/well for 96-well plates and 600 μL/well for 24-well plates and allowed to grow to approximately 85% confluence before used for assays. The 96-well plates were used for multiplicity of infection (MOI) assay, whereas the 24-well plates were used for the invasion assays. For the qRT-PCR and apoptosis, assays cells were seeded at 4 × 10⁸cells/mL in 12-well plates. The entire experiment was performed in triplicate on two independent occasions.

For each batch, both thighbones were collected from 30 day-of-hatch chicks per line. Each thighbone was split and bone marrow was scraped from the split thighbone using a sharp blade. The bone marrows were rinsed with 1 × PBS (Gibco), then pooled within line within each of the 3 batches, resulting in a total of 6 pooled bone marrow samples. Each pooled sample was then dissociated using 0.05% TE (Gibco) at 39 °C for 20 min. Subsequently, 3 × 10⁵ of the dissociated cells were cultured per well in multiple 6-well culture plates with 2 mL of DMEM (Gibco) with 10% FBS (Hyclone), 5% chicken serum (Gibco) and 1% antibiotics (1 × Antibiotic-Antimycotic, Gibco) at 39 °C with 5% CO2 until passage 2 (P2). For passaging each cell batch, BMCs from a single well were split into 4 wells in every subculture. BMCs were frozen in liquid nitrogen at P2 until treatments were performed. Before treatments, equal of BMCs from all three batches were mixed, and then 5 × 10⁵ of the mixed batch cells were cultured until P3. The cell viability, determined by trypan blue staining, was over 95% in every subculture.

Procedures for isolating and culturing ESCs, PGCs, and SSCs were performed as previously reported (Zhang Z. et al., 2015 (link)). The culturing medium generally contained 43.5 ml of Knockout-DMEM (Gibco, New York, NY, United States, 10829018), 100 μl of gentamicin (Solarbio, Beijing, China, G8170), 0.2 μl of β-mercaptoethanol (Sigma, Missouri, United States, M3148), 200 μl of non-essential amino acids (Sigma, Missouri, United States, M7145), 1 ml of chicken serum (Gibco, New York, NY, United States, 16110-082), 100 μl of SCF (Sigma, Missouri, United States, 300-07-10), 100 μl of bFGF (Sigma, Missouri, United States, F0291), 50 μl of LIF (Millipore, MA, United States, ESG1106), and 500 μl of penicillin (Solarbio, Beijing, China, P1400-100). The detailed constituents in supplemented media for culturing ESCs, PGCs, and SSCs are shown in Supplementary Table S1.

DT40 cells were grown in RPMI medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Labtech), 1% chicken serum (Gibco) and 100 U µl⁻¹ penicillin/streptomycin at 39°C, 5% CO₂. To arrest cells in mitosis, cultures were treated for 12–14 h with 0.5 µg ml⁻¹ nocodazole (Sigma-Aldrich). For SILAC labelling cells were grown in RPMI media (Invitrogen) with 10% (v/v) dialysed FBS (Sigma), 100 µg ml⁻¹ U-13C615N2-l-lysine : 2HCl and 30 µg ml⁻¹ U-13C615N4-l-arginine : HCl (Sigma) for six cell cycles.
To measure the mitotic index, cells were adhered to Polysine slides (VWR). Slides were incubated in 75 mM KCl for 4 min at room temperature and fixed in 75% methanol/25% acetic acid. The slides were inspected under a light microscope and 1000 cells were counted for each condition.