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| Status |
Public on Apr 26, 2022 |
| Title |
E11.5 Endothelial Cells (Reaggregate; 96Hrs) rep2 |
| Sample type |
SRA |
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| Source name |
Embryo derived and then Re-aggregate cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: Endothelial Cells
surface marker: b2-MG-GFP+/PDGFRA-/PDGFRB-/CD31+/VE-Cadherin+/CD45-/CD41-
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| Treatment protocol |
Cells were extracted as either freshly embryo-derived or from co-aggregate cultures.
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| Growth protocol |
E11.5- and E13.5 AGM PSCs were isolated from Mesp1DsRed and Wnt1DsRed embryos and cultured as previously described1, 2. Dissected AGMs were transferred into collagenase Type II (263 U/ml; Worthington Biosciences, Lakewood, NJ) and placed on a shaker at 37˚C for 15 min. The supernatant was passed through a 40 µm filter into a fresh tube and inactivated with 100% FCS. Cells were washed twice in 2% FCS in PBS and plated in MEM (Invitrogen, Carlsbad, CA) with 20% FCS, and penicillin/ streptomycin/ glutamine (P/S/G, Invitrogen)2 and cultured in the incubator at 37˚C, 5% CO2 for 72 h. At the end of 72 hrs, cells were washed in PBS to remove non-adherent cells and cultures were continued in fresh medium. Cells were passaged on reaching 80% confluence. After passaging, cells were placed back in tissue culture flasks with MEM + 20% FCS + P/S/G for bulk passaging. Cells were routinely cryopreserved in 10% DMSO and 90% culture medium. Re-aggregates were made by reconstituting FAC sorted cells from one AGM equivalent or 50,000 adult cardiac endothelial cells with 200,000 AGM PSCs. Dissociated cells were resuspended in 10μl of IMDM+ (IMDM Invitrogen) containing 20% fetal calf serum, 4mM L-glutamine, 50units/ml penicillin/streptomycin, 0.1mM mercaptoethanol, 100ng/ml IL, 100ng/ml SCF and 100ng/ml Flt3L (Peprotech). Re-aggregates were made by centrifugation in a yellow tip occluded by parafilm at 300 x g for 5 min and cultured on top of a 0.65μm Durapore filter (Millipore, Cat. No. DVPP02500) at the gas-liquid interface as described3. Tissues were maintained in 5% CO2 at 37C in a humidified incubator.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 200 ng of total RNA for the construction of sequencing libraries.
RNA libraries were prepared by Novogene (Hong Kong) for sequencing using standard Illumina protocols
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
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| Description |
Re-aggregate cells
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| Data processing |
Raw sequencing reads filtered for adapter contamination, low quality scores, and excluded reads in which more than 10% of bases were unknown.
Filtered paired-end RNA-seq reads aligned to the mouse genome (mm10) using the software package STAR with standard parameters.
Genome_build: mm10
Supplementary_files_format_and_content: bigwig files for visualization on the UCSC genome browser
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| Submission date |
May 15, 2018 |
| Last update date |
Apr 26, 2022 |
| Contact name |
Yizhou Huang |
| E-mail(s) |
yizhou.huang1@unsw.edu.au
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| Organization name |
University of New South Wales
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| Street address |
High Street
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| City |
UNSW Sydney |
| State/province |
NSW |
| ZIP/Postal code |
2052 |
| Country |
Australia |
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| Platform ID |
GPL21273 |
| Series (1) |
| GSE114464 |
Mesoderm-derived PDGFRA+ cells regulate emergence of hematopoietic stem cells in the dorsal aorta |
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| Relations |
| BioSample |
SAMN09210211 |
| SRA |
SRX4083885 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3142275_V30.bw |
41.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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