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| Status |
Public on Nov 01, 2020 |
| Title |
scRNA_WT_D4 |
| Sample type |
SRA |
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| Source name |
Day 4 Flk-1hi/PdgfRa- EB cells
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| Organism |
Mus musculus |
| Characteristics |
strain: 129/Sv//Ev
tissue: hematovascular mesoderm
genotype: wildtype
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| Treatment protocol |
NA
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| Growth protocol |
WT (CCE) and Eomes-null (CCE, clone 6A6) ESC lines were maintained in feeder free culture conditions in serum + LIF as previously described(Costello et al., 2011). 48-72 hrs prior to induction of hematopoietic differentiation cells were washed with PBS and cultured in serum free ES media containing 50% Neurobasal Media (Gibco, Cat # 21103049), 50% DMEM/F12 (Gibco, Cat # 11320033), supplemented with 0.5X of both N2 (Gibco, Cat#17502048) and B27 (Gibco, Cat# 17504044), 1%Pen/Strep, 1% glutamine, 0.05% BSA (Gibco, Cat# 15260037), 1 uM PD0325091, 3 uM CHIR99021 and 1000 U/ml LIF. At D0 cells were dissociated using Trypsin-LE (Gibco) and seeded at a density of 1×10^5 cells ml in serum-free differentiation (SF-D) media(Nostro et al., 2008) and cultured on an orbital shaker at 70 rpm for ~18 hrs in the absence of growth factors to form EBs. At D2, EBs were split 1:3 in SF-D media containing recombinant human (rh) VEGF (5 ng ml-1; R&D Systems), rhBMP4 (10 ng ml-1; R&D Systems) and Activin A (5 ng ml-1; R&D Systems) for 48 hr. At D4, EBs were split 1:2 in SF-D media containing rhVEGF (5 ng ml-1; R&D Systems), rhBMP4 (10 ng ml-1; R&D Systems) and Activin A (5 ng ml-1; R&D Systems) for 24 hr.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Processing of samples was performed according to the 10x protocol using the Chromium Single Cell 3’ library & Gel Bead Kit v3 (10x Genomics).
Barcoded cell cDNAs were pooled and converted to sequence ready libraries. Multiplexed libraries were then sequenced on Illumina Nextseq using a high output 150bp Nextseq V2.5 kit.
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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 |
Sequencing data was demultiplexed in the binary base call (BCL) format, FASTQ files were aligned to the mm10 genome using 10X Genomics Cell Ranger software (version 2.0.0) and unique molecular identifier (UMI) counts were determined.
The Seurat v3 software package(Butler et al., 2018; Stuart et al., 2019) was used in R Studio to perform scRNA-Seq analysis.
Pre-processing was performed and cells with > 2000 RNA features and < 8 % mitochondrial RNA were used for downstream analysis.
Genome_build: mm10
Supplementary_files_format_and_content: [.tsv] barcode and feature files [.mtx] matrix files
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| Submission date |
Nov 07, 2019 |
| Last update date |
Nov 02, 2020 |
| Contact name |
Luke Thomas Gilbert Harland |
| E-mail(s) |
luke.harland@path.ox.ac.uk
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| Organization name |
Oxford University
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| Department |
Sir William Dunn School of Pathology
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| Lab |
Elizabeth Robertson
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| Street address |
South Parks Road
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| City |
Oxford |
| ZIP/Postal code |
OX1 3RE |
| Country |
United Kingdom |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE140005 |
The T-box Transcription Factor Eomesodermin Governs Hemogenic Competence of Yolk Sac Mesodermal Progenitors |
| GSE140061 |
The T-box Transcription Factor Eomesodermin Governs Hemogenic Competence of Yolk Sac Mesodermal Progenitors [scRNA-Seq] |
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| Relations |
| BioSample |
SAMN13229261 |
| SRA |
SRX7114594 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4153428_scRNA_WT_D4_barcodes.tsv.gz |
21.6 Kb |
(ftp)(http) |
TSV |
| GSM4153428_scRNA_WT_D4_features.tsv.gz |
272.8 Kb |
(ftp)(http) |
TSV |
| GSM4153428_scRNA_WT_D4_matrix.mtx.gz |
66.3 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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