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| Status |
Public on Jul 08, 2024 |
| Title |
Bmp4_explant_10h |
| Sample type |
SRA |
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| Source name |
embryonic
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| Organism |
Danio rerio |
| Characteristics |
tissue: embryonic
cell line: zebrafish embryonic stem cells
cell type: stem cell
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| Treatment protocol |
To generate bmp4 explants, bmp4 (8-10 pg) mRNA was injected into one animal pole blastomere at the 128-cell stage. Then the animal pole region was explanted and raised to 24hpf. To generate BMP4 induced human embryoids, human embryonic stem cells (hESCs) of the H9 line were grown on Matrigel-coated 12-well plates for a period ranging from 48 to 72 hours, utilizing them when they reached an approximate confluence of 70%. On the initial day of the experiment, both wild-type H9 cells and ihBmp4 cells were treated with accutase to facilitate their dissociation. They were then transferred to 96-well U-bottom low attachment plates for culture. The seeding density was adjusted to 1300 cells per well for H9 cells and 400 cells per well for ihBmp4 cells, using 50 μl of an aggregation medium based on TeSR-E6 and supplemented with the ROCK inhibitor 10μM Y27632. After a 24-hour incubation period, both types of cells formed embryoid bodies (EBs) in their respective wells. These H9 and ihBmp4 EBs were then co-cultured in the same well, with 80 μl of the TeSR-E6 based medium to encourage fusion. Between days 2 and 4 of the culture period, the EBs were subjected to an induction process to enhance Bmp4 expression, which initiated the morphogenesis phase. The culture medium was replaced with a fresh TeSR-E6 medium that included 75 ng ml-1 Doxycycline and 50 ng ml-1 FGF2 to stimulate these processes. Starting from day 4, the Doxycycline and FGF2 were omitted from the culture medium. The EBs were then maintained in a TeSR-E6 based medium for continued growth and development.
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| Growth protocol |
Bmp4 explants were cultured in Dulbecco's modified Eagle’s medium (DMEM/F-12 with 10 mM HEPES, 1x MEM with non-essential amino acids, 7 mM CaCl2, 1 mM sodium pyruvate, 50 μg/ml gentamycin, 100 μM 2-mercaptoethanol, 1× antibiotic-antimycotic ,10% serum replacement) and kept at 28.5℃.BMP4 induced human embryoids were cultured in a 96-well U-shaped ultra-low plate with culture medium (TeSR-E6 medium supplemented with 0.5% N2 and 0.5% B27 to support cell growth). Small molecule compounds and recombinant proteins inducing differentiation were added according to the treatment protocol. Samples were collected on day 5 and 6 of differentiation.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Single-cell RNA of each sample was extracted by following the Chromium Controller and Chromium Single Cell 30 Library & Gel Bead Kit v3.1(10x Genomics, PN-1000121) protocol.
Libraries were prepared using Single Cell 3′Library & gel Bead kit v3.1 (10x Genomics, Cat# PN-1000121) according to the manufacturer’s protocol for 10000 cell recovery.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
10x Genomics
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| Data processing |
Illumina sequencing reads were aligned to the zebrafish (GRCz11) or human mRNA reference genome (GRCh38) using the 10x Genomics CellRanger pipeline (version 3.0.2) with default parameters.
Assembly: GRCz11, GRCh38
Supplementary files format and content: tar archive includes filtered_gene_bc_matrices after running CellRanger pipeline
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| Submission date |
Jun 27, 2024 |
| Last update date |
Jul 08, 2024 |
| Contact name |
Tao cheng |
| E-mail(s) |
11818118@zju.edu.cn
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| Phone |
0571-88208276
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| Organization name |
Zhejiang University
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| Department |
Institute of genetics
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| Street address |
886 Yuhangtang Road
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| City |
Hang Zhou |
| State/province |
Zhe Jiang |
| ZIP/Postal code |
310000 |
| Country |
China |
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| Platform ID |
GPL24995 |
| Series (1) |
| GSE270989 |
in vitro construction of an embryonic caudal organizer |
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| Relations |
| BioSample |
SAMN42147537 |
| SRA |
SRX25138329 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8366752_Bmp4_explant_10h_filtered_feature_bc_matrix.tar.gz |
101.0 Mb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
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