ATAC-seq of Mus musculus embryonic cell types in response to Pbx1 knockout
ATAC-seq of Mus musculus embryonic cell types in response to Pbx1 knockout
For all conditions, two biological replicate samples were collected from independent experiments. EpiSC lines derived from E6.5 mouse embryos (129S6/SvEvTac background) were a kind gift from Dr. M.M. Rothová (The Novo Nordisk Foundation Center for Stem Cell Biology, University of Copenhagen, Copenhagen, Denmark). The lines were derived in EpiSC medium as previously described [1]. EpiSC lines were maintained in 6-well cell-culture plates (Corning, 3506) pre-coated with 15 µg/ml human Fibronectin (Merck Millipore, FC01015) in N2B27 medium (prepared as described in Ying and Smith, 2003) supplemented with 20 ng/ml Activin A, 10 ng/ml bFGF and 1 µM XAV939 (EpiSC medium). Both mESC lines were cultured in 0.1% gelatin-coated plates 6-well Corning cell-culture plates. mESCs were cultured in standard conditions in either serum/LIF or 2i/LIF conditions with CHIR-99021, PD03 and LIF (made in house). All lines used in this study were karyotyped and routinely tested for mycoplasma.   [1] Guo, G. et al. Klf4 reverts developmentally programmed restriction of ground state pluripotency. Development 136, 1063-9 (2009). Generation of Pbx1 ESC/EpiSC lines using the CRISPR/Cas9 system. The strategy to inactivate Pbx1 in ESCs and EpiSCs was the same. Pbx1-KO lines were generated by inserting a floxed CMV-hygromycin-Poly (A) cassette between Exon 2 and Intron 2 of Pbx1 to disrupt Pbx1 gene expression, employing the CRISPR Nickase system. Guide RNAs (gRNAs) were designed using the CRISPR/Cas9 design tool (crispr.mit.edu) and cloned into pX335 (a gift from Prof. F. Zhang; Addgene, 42335). Cells were co-transfected with a pair of gRNA-pX335 plasmids (gRNA 1: GTCCAGGCTCTGGTCCGTGA; gRNA 2: CCAAGTGAGTGCCCCCACCC; 100 ng each vector) and 1 µg linearized targeting vector using Lipofectamine 2000 (Thermo Fisher Scientific, 11668030), seeded at low density and selected for hygromycin resistance for 8-10 days. Single colonies were screened by diagnostic PCR, Southern blot and Western blot analyses. Pbx1 gRNA used (GGCCATCCCGACCCCAGCGT)  EpiSC differentiation to germ-layer lineages. All differentiation was conducted in serum-free, feeder-free and monolayer conditions in chemically-defined N2B27-based media. EpiSCs were seeded at ~10.5 x 103 cells/cm2 in 6-well cell-culture plates pre-coated with 15 µg/ml human Fibronectin, and cultured in EpiSC medium for 24h. EpiSCs were differentiated towards either APSD using 30 ng/ml Activin A, 4 µM CHIR-99021, 20 ng/ml bFGF and 100 nM PIK-90 (APS medium) for 24 For NMP/PSM differentiation, EpiSCs were first differentiated towards APSD, and then to NMP/PSM using 1 µM A-83-01, 3 µM CHIR-99021, 250 nM LDN193189 and 20 ng/ml bFGF for 48-72h (PSM media). For DE differentiation, EpiSCs were first differentiated towards APSD in N2B27-basal media over 24 h, and then to DE using 30 ng/ml Activin A, 4 µM CHIR-99021 and 100 nM PIK-90 in RPMI(Glutamax) + B27 (Without insulin) basal media (DE medium) for 48 h. EpiSCs were differentiated towards neuroectoderm based on known media requirements [1]. Differentiation of EpiSCs towards PPS and ExM were done as previously described [2].     [1] Greber, B. et al. Conserved and divergent roles of FGF signaling in mouse epiblast stem cells and human embryonic stem cells. Cell Stem Cell 6, 215-26 (2010).  [2] Drozd, A.M. et al. Progesterone Receptor Modulates Extraembryonic Mesoderm and Cardiac Progenitor Specification during Mouse Gastrulation. Int J Mol Sci 23(2022). Nuclear extracts (NE) for EMSA were prepared from in vitro-differentiated paraxial mesoderm cells. Cells were resuspended in 200 μl lysis buffer (10 mM HEPES pH 7.9, 30 mM KCl, 1.5 mM MgCl2, 1 mM DTT, supplemented with 1x Protease inhibitor cocktail (Roche, 11836170001)), kept on ice for 10 min and supplemented with addition of Triton X-100 to a final concentration of 0.1%. The nuclei were collected by centrifugation at 4 °C. Nuclear extracts were prepared by resuspending the nuclear pellet in 50 μl nuclear buffer (20 mM HEPES pH 7.9, 25% (v/v) glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 1 mM DTT, supplemented with 1x Protease inhibitor cocktail). NE preparations were incubated at 4 °C with rotation for 30 min and cleared by centrifugation. Finally, the extracts were quantified by NanoDrop, aliquoted and stored at −80 °C. ATAC-seq was performed on 50,000 cells per replicate as described in Buenrostro et al.64 (with modifications based on Corces et al.65), on EpiSCs and PSM-differentiated cell populations at desired time-points. Libraries were generated using the Ad1_noMX and Ad2.1–2.16 barcoded primers64 and amplified for 10 total PCR cycles. Libraries were purified with AMPure XP beads to remove contaminating primer dimers and fragments >1,000 bp. Library quality was assessed using the Fragment analyzer and quantitated by Qubit assay. The libraries were sequenced with 50 bp paired-end reads on a Next-Seq 500 Sequencer (Illumina) at the DanStem Genomics Platform (University of Copenhagen, Copenhagen, Denmark).