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| Status |
Public on Jan 24, 2021 |
| Title |
human blastoids, scRNA-Seq |
| Sample type |
SRA |
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| Source name |
human blastoids
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| Organism |
Homo sapiens |
| Characteristics |
tissue: blastoids
internal sample: LW36
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| Growth protocol |
For the generation of human blasatoids, 60~70% confluent naïve hPSCs were dissociated into single cells by incubation with Tryple Express (GIBCO) at 37°C for 3 min. Then, the cell resuspension was pre-incubated at 37 °C in 5% CO2 on gelatinized tissue culture plates for 30 min to remove MEF cells. The supernatant containing the naïve hPSCs cells were collected, passed through a 40 ?m cell strainer, and counted. Meanwhile, the AggreWell?400 (STEMCELL Technologies) was prepared according to manufacturer?s instructions. Briefly, wells were rinsed with anti-adhesion rinsing solution (STEMCELL Technologies) then centrifuged at 2,000g for 5 min and incubated at room temperature for 10 min. After incubation, wells were washed with naïve hPSC medium once and 0.5 mL fresh naïve hPSC medium containing 5 ?M Y-27632 was added (Selleckchem). Approximately 30,000 cells (~25 cells / microwell for 5iLA-cultured WIRB3 hESCs. For other cell lines, starting cell number need to be optimized) were resuspended in 1mL 5iLA medium supplemented with 5? M Y-27632 and seeded into one well of prepared AggreWell?400 24-well plate. The plate was centrifuged at 200 g for 1 min and placed in a 37 °C and 5% CO2 incubator. Aggregates formed following ~12 hours culture, and the medium was replaced with HDM or TDM. The first day of changing to differentiation medium (HDM or TDM was designated as day 1. Naïve hPSC medium is first changed to TDM (day 1). Fresh TDM is replaced every 2 days until the cavity structures appear (usually it takes 6-7 days for 5iLA-WIRB3-hESCs, and the time varies among different cell lines, different starting culture conditions, different starting cell numbers and sometimes different batches), then the medium is replaced with HDM and cultured for 1-2 days.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Single cell suspension was washed with 1X PBS (0.04% BSA) before 10x Genomics procedure. The concentration of single cell suspension was adjusted to about 500 to 1000 cells/µL and was loaded on the 10x Genomics’ Chromium™ system (10x Genomics, Pleasanton, CA) with the aim of 6000 to 10000 cells per channel (Chromium Next GEM Single Cell 3ʹ v3.1, catalog number 1000121).
Single cell RNA sequencing libraries were constructed following the manufacturer’s instructions (Zheng et al., 2017).
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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 |
BCL files generated by Illumina sequencing systems were demultiplexed and converted to standard FASTQ files using mkfastq function from Cell Ranger pipeline (version 4.0.0).
Reads were processed to generate expression (UMI count) matrix using Cell Ranger pipeline (version 4.0.0) with default parameters, except the parameter of expected number of cells, which was adjusted based on individual experiment.
Genome_build: GRCh38
Supplementary_files_format_and_content: Raw UMI count matrix representing gene expression values (comma-separated) were generated as described above. Each column represents a single cell, each row represents a single gene.
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| Submission date |
May 14, 2020 |
| Last update date |
Jan 24, 2021 |
| Contact name |
Gary Hon |
| Organization name |
UT Southwestern
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| Department |
Department of Obstetrics and Gynecology
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| Lab |
Hon Lab
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| Street address |
5323 Harry Hines Blvd.
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| City |
Dallas |
| ZIP/Postal code |
75390 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE150570 |
Generation of blastocyst-like structures from human embryonic stem cells I [scRNA-seq] |
| GSE150578 |
Generation of blastocyst-like structures from human embryonic stem cells |
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| Relations |
| BioSample |
SAMN14924195 |
| SRA |
SRX8341669 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4552488_matrix_readcount_lw36.csv.gz |
30.7 Mb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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