ERC000011
ENA-CHECKLIST
2023-12-01T00:42:58Z
ENA-FIRST-PUBLIC
2023-12-01T00:42:58Z
ENA-LAST-UPDATE
SAMEA114540915
External Id
reNEW, Novo Nordisk Foundation Centre of Stem cell Medicine, University of Copenhagen
INSDC center name
2023-12-01T00:42:58Z
INSDC first public
2023-12-01T00:42:58Z
INSDC last update
public
INSDC status
ERS16522244
SRA accession
E-MTAB-13407:Pbx1KO_clone15_ExM_rep1
Submitter Id
ArrayExpress
broker name
ExM
cell_type
not collected
collection date
house mouse
common name
CRISPR/Cas9 + Pbx1KO gDNA
crispr/cas9 treatment
Protocols: 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).
description
embryo
developmental stage
Unsorted
flow cytometry processing
CRISPR/Cas9 mediated Pbx1 homozygous knockout
genotype
not collected
geographic location (country and/or sea)
EpiSCs + 24h towards PPS + 24h towards ExM
growth condition
NB501779
illumina sequencing machine
Extracted from post-implantation embryo and differentiated
implantation stage at sampling
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
rep1
replicate
Mus musculus
scientific_name
9/16/2019
sequencing date
AHCHVMBGXC
sequencing kit barcode
129S6/SvEvTac
strain
Pbx1KO_clone15_ExM_rep1
title