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| Status |
Public on Dec 29, 2019 |
| Title |
ucla1t2c.PGCLCd3 |
| Sample type |
SRA |
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| Source name |
hESCs
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| Organism |
Homo sapiens |
| Characteristics |
genotype: TFAP2C mutant
cell type: hESCs
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| Treatment protocol |
hESCs were dissociated into single cells with 0.05% Trypsin-EDTA (GIBCO, 25300-054) and plated onto Human Plasma Fibronectin (Invitrogen, 33016-015)-coated 12-well-plates at the density of 200,000 cells/well in 2mL/well of iMeLC media, which is composed of 15% KSR (GIBCO, 10828-028), 1x NEAA (GIBCO, 11140-050), 0.1mM 2-Mercaptoethanol (GIBCO, 21985-023), 1x Penicillin-Streptomycin-Glutamine (GIBCO, 10378-016), 1mM sodium pyruvate (GIBCO, 11360-070), 50ng/mL Activin A (Peprotech, AF-120-14E), 3mM CHIR99021 (Stemgent,04-0004), 10mM of ROCKi (Y27632, Stemgent, 04-0012-10), and 50ng/mL primocin in Glasgow’s MEM (GMEM) (GIBCO, 11710-035). After 24 hr, iMeLCs were dissociated into single cells with 0.05% Trypsin-EDTA and plated into ultra-low cell attachment U-bottom 96-well plates (Corning, 7007) at the density of 3,000 cells/well in 200ml/well of hPGCLC media, which is composed of 15% KSR (GIBCO, 10828-028), 1x NEAA (GIBCO, 11140-050), 0.1mM 2-Mercaptoethanol (GIBCO, 21985-023), 1x Penicillin-Streptomycin-Glutamine (GIBCO, 10378-016), 1mM sodium pyruvate (GIBCO, 11360-070), 10ng/mL human LIF (Millipore, LIF1005), 200ng/mL hu- man BMP4 (R&D systems, 314-BP), 50ng/mL human EGF (R&D systems, 236-EG), 10mM of ROCKi (Y27632, Stemgent, 04-0012-10), and 50ng/mL primocin in Glasgow’s MEM (GMEM) (GIBCO, 11710-035).
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| Growth protocol |
hESCs were cultured on mitomycin C-inactivated mouse embryonic fibroblasts (MEFs) in hESC media, which is composed of 20% knockout serum replacement (KSR) (GIBCO, 10828-028), 100mM L-Glutamine (GIBCO, 25030-081), 1x MEM Non-Essential Amino Acids (NEAA) (GIBCO, 11140-050), 55mM 2-Mercaptoethanol (GIBCO, 21985-023), 10ng/mL recombinant human FGF basic (R&D systems, 233-FB), 1x Penicillin-Streptomycin (GIBCO, 15140-122), and 50ng/mL primocin (InvivoGen, ant-pm-2) in DMEM/F12 media (GIBCO, 11330-032). The hESCs were split every 7 days using Collagenase type IV (GIBCO, 17104-019). All hESC lines used in this study are registered with the National Institute of Health Human Embryonic Stem Cell Registry and are available for research use with NIH funds. Mycoplasma test (Lonza, LT07-418) was performed every month. All experiments were approved by the UCLA Embryonic Stem Cell Research Oversight Committee.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Single cell suspension was made from hESCs, iMeLCs, day 1 to day 4 aggregates after trypsin treatment. Single cells were washed three to five times in PBS supplemented with 0.04% BSA. Single cell suspension was sequenced using 10X Genomics.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
The reads from single cell RNA-seq were aligned to the GRCh38 human reference genome using the Cell Range v.2.2.0 pipeline with default parameters (10x Genomes).
The generated cell-by-gene unique molecular identifier (UMI) count matrix was analyzed using the Seurat R package v.2.3.4.
We used Seurat(v3.0.0) package for further analysis and only kept the cells expressing at least 200 genes and genes with expression in at least 3 cells. The cells were also filtered by the maximum of 8000 expressed genes and of 20% mitochondrial genes.
The UMI counts were then normalized for each cell by the total expression, multiplied by 10000, and log-transformed.
Genome_build: GRCh38
Supplementary_files_format_and_content: txt files with raw read counts
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| Submission date |
Dec 04, 2019 |
| Last update date |
Dec 29, 2019 |
| Contact name |
Manolis Kellis |
| Organization name |
MIT
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| Department |
EECS
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| Street address |
32 Vassar St. D524
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE140021 |
Human Primordial Germ Cells are Specified from Lineage Primed Progenitors |
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| Relations |
| BioSample |
SAMN13480878 |
| SRA |
SRX7267449 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4202937_ucla1t2c.PGCLCd3.rawmatrix.txt.gz |
9.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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