KEY RESOURCES TABLES
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Anti-CDX-2, Clone CDX2-88 antibody | BioGenex | AM392-5M, RRID: AB_2650531 |
| Human SOX17 Affinity Purified Polyclonal Ab antibody | R&D Systems | AF1924, RRID: AB_355060 |
| Sox-2 (E-4) antibody | Santa Cruz Biotechnology | sc-365823, RRID: AB_10842165 |
| Anti-Human SOX2 | BioGenex | AN833-5M, RRID: not available |
| GATA3-human antibody | Santa Cruz Biotechnology | sc-268, RRID: AB_2108591 |
| Keratin 18 | Sigma | SAB4501665, RRID: AB_10746153 |
| Anti-ZO-1 Monoclonal Antibody | Innovate Research | 33-9100, RRID: AB_87181 |
| Alexa Fluor® 594 Phalloidin antibody | Invitrogen | A12381, RRID: AB_2315633 |
| Phospho-Stat3 (Tyr705) (D3A7) | Cell Signaling Technology | 9145T, RRID: not available |
| Donkey anti-rabbit IgG (H+L) Antibody, Alexa Fluor™ 647 | Invitrogen | A31573, RRID: AB_2536183 |
| Donkey anti-mouse IgG (H+L) Antibody, Alexa Fluor™488 | Invitrogen | A21202, RRID: AB_141607 |
| Donkey anti-Goat IgG (H+L) Antibody, Alexa Fluor™ 555 | Invitrogen | A-21432, RRID: AB_2535853 |
| Chemicals, Peptides, and Recombinant Proteins | ||
| N2 supplement (100X) | Gibco | Cat. No. 17502-048 |
| B27 supplement (50X) | Gibco | Cat. No. 17504-044 |
| Insulin-Transferrin-Selenium-Ethanolamine (ITS-X) | Gibco | Cat. No. 51500-056 |
| Recombinant Human LIF | Peprotech | Cat. No. 300-05 |
| CHIR-99021 | Selleckchem | Cat. No. S1263 |
| PD0325901 | Selleckchem | Cat. No. S1036 |
| Recombinant Human FGF-basic | Peprotech | Cat. No. 100-18B |
| Recombinant Human/Murine/Rat Activin A | Peprotech | Cat. No. 120-14E |
| Emricasan | Selleckchem | Cat. No. 50-136-5234 |
| Polyamine supplement 5ml | Sigma-Aldrich | Cat. No. P8483 |
| Antioxidant supplement, 5ml | Sigma-Aldrich | Cat. No. A1345 |
| trans-ISRIB,10mg | Tocris | Cat. No. 5284 |
| Chroman 1 (HY15392), 5mg | Medchemexpress | Cat. No. 502029121 |
| mTeSR™ Plus | STEMCELL Technologies | Cat. No. 100-1130 |
| WH-4-023 | Tocris | Cat. No. 5413 |
| endo-IWR 1 | Tocris | Cat. No. 3532 |
| XAV-939 | Tocris | Cat. No. 3748 |
| L-Ascorbic acid 2-phosphate | Sigma-Aldrich | Cat. No. A8960 |
| AlbumiNZ Free Fatty Acid | MP Biomedicals | Cat. No. 199899 |
| Dulbecco’s phosphate buffered saline (1X), no calcium, no magnesium (DPBS) | Corning | Cat. No. 354277 |
| DMEM/F12 | Gibco | Cat. No. 11320-033 |
| GlutaMAX (100X) | Gibco, Cat. No. 35050-061 | Cat. No. 35050-061 |
| MEM Non-Essential Amino Acids (100X) | Gibco | Cat. No. 11140-050 |
| 2-Mercaptoethanol (1000X) | Gibco | Cat. No. 21985-023 |
| Fetal Bovine Serum, heat inactivated | Gibco | Cat. No. A3840001 |
| Deposited Data | ||
| 8 cell and 16 cell | Single-cell RNA sequencing reveals developmental heterogeneity of blastomeres during major genome activation in bovine embryos30 | GSE99210 |
| Zygote, 2 cell, 8 cell, morula and blastocyst | Reprogramming barriers in bovine cells nuclear transfer revealed by single-cell RNA-seq analysis29 | PRJNA727165 |
| gastrulation embryos | Provided by Dr. Peter L. Pfeffer, Gene expression analysis of bovine embryonic disc, trophoblast and parietal hypoblast at the start of gastrulation31 | GSE215409 |
| In vivo blastocyst and in vitro blastocyst | Provided by Dr. Zongliang Jiang | GSE215409 |
| Blastoid | This paper | GSE221248 |
| Software and Algorithms | ||
| Cell Ranger (v 3.1.0) | www.10xgenomics.com | https://github.com/10XGenomics/cellranger; RRID:SCR_017344 |
| Seurat (v 3.1.4) | Stuart T, Butler A, Hoffman P, et al. Comprehensive integration of single-cell data. Cell. 2019;177(7):1888–1902. e1821. | https://satijalab.org/seurat/articles/get_started.html; RRID:SCR_016341 |
| Trim Galore (v 0.6.4) | www.bioinformatics.babraham.ac.uk/projects/trim_galore/ | http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/; RRID:SCR_011847 |
| HISAT2 (v 2.1.0) | Kim D, Paggi JM, Park C, Bennett C, Salzberg SL. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nature biotechnology. 2019;37(8):907–915. | http://daehwankimlab.github.io/hisat2/; RRID:SCR_015530 |
| HTSeq-count (v 2.0.2) | Putri GH, Anders S, Pyl PT, Pimanda JE, Zanini F. Analysing high-throughput sequencing data in Python with HTSeq 2.0. Bioinformatics. 2022;38(10):2943–2945. | https://htseq.readthedocs.io/en/master/htseqcount.html; RRID:SCR 011867 |
| clusterProfiler (v 3.14.3) | Wu T, Hu E, Xu S, et al. clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. The Innovation. 2021;2(3):100141. | http://bioconductor.org/packages/release/bioc/html/clusterProfiler.html; RRID:SCR 016884 |
| Monocle3 (v 0.2.3.0) | Cao J, Spielmann M, Qiu X, et al. The single-cell transcriptional landscape of mammalian organogenesis. Nature. 2019;566(7745):496–502. | https://cole-trapnell-lab.github.io/monocle3/; RRID:SCR 018685 |
| Scanpy (v 1.9.3) | Wolf, F., Angerer, P., Theis, F. SCANPY: large-scale single-cell gene expression data analysis. Genome Biol. 2018;19(15). | https://github.com/scverse/scanpy |
| Scikit-learn (v 1.2.1) | Pedregosa, Fabian, et al. Scikit-learn: Machine learning in Python. the Journal of machine Learning research. 2011;12:2825–2830. | https://scikit-learn.org/stable/ |
| Imaris XT | Oxford Instruments | https://imaris.oxinst.com/products/imaris-for-cell-biologists |
| Code used in this study |
https://github.com/anaorsi/CSC_BovineBlastoids/tree/CSC
https://zenodo.org/record/7792885#.ZCnFqi-B2Ak |
DOI: 10.5281/zenodo.7792885 |
| Other | ||
| AggreWell 400 | STEMCELL Technologies | Cat. No. 34,415 |
| Anti-Adherence Rinsing | STEMCELL Technologies | Cat. No. 07,010 |
| Dispase | STEMCELL Technologies | Cat. No. 07913 |
| Accumax | Thermo Fisher | Cat. No. 00-4666-56 |
| TrypLE™ Express | Gibco | Cat. No. 12605036 |
| ProFreeze Freezing medium | Lonza, 12-769E | Cat. No. 12-769E |
| CoolCell | Corning | Cat. No. 432000 |
| PureCoat™ Amine 6 Well Plate | Corning | Cat. No. 354721 |
| BD LSR II Flow cytometer | BD Biosciences | N/A |
| Nikon CSU-W1 spinning-disk super resolution by optical pixel reassignment (SoRa) | Nikon | N/A |
| Chromium Next GEM Single Cell 3’ Kit v3.1 | 10X Genomics | Cat. No. 1000269 |
| Chromium Next GEM Chip G Single Cell Kit | 10X Genomics | Cat. No. 1000127 |
| Chromium Controller & Next GEM Accessory Kit | 10X Genomics | Cat. No. 000204 |
| Centrifuge HERMLE Z 326 K | HERMLE Labortechnik GmbH | Z 326 K |
| Stereo Microscope SMZ800N | Nikon | SMZ800N |
| Thermo Plate | TOKAI HIT | N/A |
| Micropipette Puller P-97 | Sutter Instruments | N/A |
| Micro Forge MF-900 | Narishige | N/A |
| ECHO Revolve | Echo | N/A |
| Nikon Eclipse Ts2 Inverted microscope | Nikon | N/A |
| Hypoxic humidified Incubator | NuAire | N/A |
| CO2 humidified Incubator | NuAire | N/A |
| 8 well μ-siles | Ibidi | Cat. No. 80821 |