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| Status |
Public on Oct 17, 2019 |
| Title |
Blastocysts |
| Sample type |
SRA |
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| Source name |
Blastocysts
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| Organism |
Mus musculus |
| Characteristics |
sample type: embryos
sample source: Blastocysts
Sex: pooled male and female
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| Growth protocol |
EPS colonies were dissociated into single cells by incubation with TrypLE (Gibco). To remove irradiated MEF cells, cell resuspension was transferred into a 0.1% gelatin-coated plate and incubated at 37°C for 30 min. EPS cells were collected, filtered through a 40 μm cell strainer, and counted. AggreWell 400 (STEMCELL Technology) were prepared following the manufacturer’s instructions. The basal medium for the EPS-blastoid induction medium is composed of 25% TSC basal medium, 25% N2B27 basal medium, and 50% KSOM (homemade or Millipore, MR020P5D). Approximately 6,000 cells (five cells per microwell for 1200 microwells) were resuspended in basal medium supplemented with 2μM ROCK inhibitor Y-27632 (Reagents Direct, 53-B80-50), 12.5ng/ml rhFGF4 (R&D, 235F4025), 12.5ng/ml Heparin (Sigma, H3149), 3μM GSK3 inhibitor CHIR99021 (Axon Medchem, 27-H76), 5ng/ml BMP4 (Proteintech, HZ-1040), and 0.5μM A83-01 (Axon Medchem, 1421) and seeded into the AggreWell 400. The plate was centrifuged at 300g for one minute and transferred into a incubator. The next day, the medium was removed and replaced with fresh induction medium without Y- 27632. Blastoids were manually picked up under a stereomicroscope for downstream experiments or analysis. For blastocysts preparation, ICR female mice were superovulated by intraperitoneal (i.p.) injection of 7.5 Unit of pregnant mares' serum gonadotrophin (PMSG) and 46–48 h later by 7.5 Unit of human chorionic gonadotrophin (HCG), and then mated with ICR male mice immediately. The blastocysts were flushed from female mice 3.5 days after detection of the vaginal plug, and cultured in KSOM under 37°C with an atmosphere of 5% CO2 in the air.
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| Extracted molecule |
total RNA |
| Extraction protocol |
EPS-blastoids were manually picked up using mouthpipette and washed three times. ~500 EPS-blastoids were harvested and dissciated with a homemade enzyme mix composed of 0.5X versene (Lonza, 17711E), 0.5X Acumax (Innovative Cell Tech, AM105), and 0.05X Dnase (STEMCELL Tech, 07900) at 37°C for 30min with agitation. Dissociated cells were spinned down and wash with PBS + 0.04%BSA for three times and resupended in the same buffer. Cell density was determined by a TC10 cell counter (Biorad, 1450001). Blastocysts were dissociated using the same protocol.
Dissociated cells (~4800 cells for EPS-blastoids and ~1000 cells for blastocysts) were loaded into the Chromium single cell controller (10xGenomics) to generate single-cell gel beads in the emulsion according to the manufacturer’s protocol. The library was generated using the Chromium Single Cell 3' Reagent Kits according to the manufacturer’s manual. The two libraries were pooled and sequenced using Nextseq 500 (150 cycles, high output).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
I1 read: contains the sample index.
R1 read: contains the cell barcode (first 16 nt) and UMI (next 10 nt).
R2 read: contains the RNA sequence.
We use STAR v2.5.1b1 to align reads to the 10x Genomics pre-built mm10 reference genome and utilized CellRanger v3.0.2 software for blastocysts (288 cells) and EPS- blastoids (3528 cells) datasets with default setting for de-multiplexing to generate feature-barcode matrix.
The R package Seurat v3.0.12 was used to read and analyze feature-barcode matrix following the steps: First, we filtered the cells that have unique feature counts over 5000 according to quality control matrix plots (184 and 2518 cells in the blastocysts and EPS-blastoids group passed the filter, respectively);
Then UMI counts were normalized with NormalizeData function using defalult settings. Seurat’s RunUMAP function was used to do non-linear dimension reduction and cluster with resolution setting as 0.2.
Differentially expressed genes in the clusters between blastocysts and EPS-blastoids were determined by the FindMarkers function using a bimodal likelihood ratio test. For the differentially expressed genes, we tested weather each had enriched GO terms in biological process and molecular functions using the ToppGene Suite3. Unsupervised clustering analysis (UCA) was performed using the R package ComplexHeatmap v2.1.0.
Genome_build: mm10
Supplementary_files_format_and_content: processed tsv files contain information on barcodes and gene annotations; the processed mtx files contain gene umi counts
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| Submission date |
Aug 12, 2019 |
| Last update date |
Oct 17, 2019 |
| Contact name |
Ronghui Li |
| E-mail(s) |
[email protected]
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| Organization name |
Salk Institute for Biological Studies
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| Department |
Gene Expression Lab
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| Lab |
Gene Expression Lab-Belmonte
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| Street address |
10010 North Torrey Pines Road
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE135701 |
Generation of Blastocyst-like Structures from Extended Pluripotent Stem Cells (single-cell RNA-Seq) |
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| Relations |
| BioSample |
SAMN12559109 |
| SRA |
SRX6697751 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4026212_Blastocyst_barcodes.tsv.gz |
20.0 Mb |
(ftp)(http) |
TSV |
| GSM4026212_Blastocyst_features.tsv.gz |
272.8 Kb |
(ftp)(http) |
TSV |
| GSM4026212_Blastocyst_matrix.mtx.gz |
18.4 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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