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| Status |
Public on Dec 23, 2014 |
| Title |
D3T3_sorted |
| Sample type |
SRA |
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| Source name |
Human Adipose Derived Stem Cells
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| Organism |
Homo sapiens |
| Characteristics |
starting material: extracted RNA
differentiation: Differentiation 3, full confluency
sorting#: FACS to tube
scrb-seq variant#: Bulk
time point: Day 3
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| Treatment protocol |
At 80% confluency (1st differentiation) or two days after confluency (2nd differentiation), the cells were induced towards an adipogenic fate by switching from growth medium to the StemPro adipogenesis differentiation medium (Life Technologies). Cells were fed every three days during the 14 days of differentiation.
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| Growth protocol |
Human adipose-derived stem cells were isolated from lipoaspirate and purified by flow-cytometry (positive CD29, CD44, CD73, CD90, CD105, CD166; negative CD14, CD31, CD45, Lin1) (Life Technology, lot 2117). Cells were grown in a 2% reduced serum medium (MesenPro RS - Life Technologies) and expanded for no more than 3 passages.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested using TrypLE Express (Life Technologies) and medium removed by pelleting the cells (5 min at 1000 rpm). RNA was immediately stabilized by resuspending the cells in RNAprotect Cell Reagent (Qiagen) and RNaseOUT Recombinant Ribonuclease Inhibitor. (1:1000, Life Technologies). Prior to sorting, cells were diluted in PBS, pH 7.4 (no calcium, no magnesium, no phenol red, Life Technologies) and stained for viability using Hoechst 33342 (Life Technologies). 384-well capture plates were filled with 5µl of diluted Phusion HF buffer (1:500, New England Biolabs). Cells were sorted individually in each well of the 384-well capture plates using a FACSAria II flow cytometer (BD Biosciences) based on Hoechst DNA staining. After sorting, plates were immediately sealed, spin down and frozen on dry ice. Sorted cells were stored at -80°C. For lipid content based single cell sorting, cells were stained with HSC LipidTOX Neutral Lipid Stain (Life Technologies) and sorted according to their relatively “high” or “low” lipid content (upper/lower 20% or 50%). For bulk SCRB-Seq, populations of both unsorted and sorted cells were lysed in QIAzol (Qiagen) and RNA was extracted and purified using Direct-zol RNA MiniPrep (Zymo Research).
Single Cell RNA Barcoding and Sequencing (SCRB-Seq), 3’ Digital Gene Expression (3’ DGE)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Differentiation 3, full confluency
D3_Bulk_UMI.dat
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| Data processing |
Mapping of read2 on mRNA refseq using BWA
Parsing the output based on cell barcode stored in read1 (first 6bp)
Gene expression computation based on Unique Molecular Identifier (UMI) counts stored in read1 (base 7 to 16)
Concatenation of single cell gene expression in gene expression matrices (processed data files)
Genome_build: hg19
Supplementary_files_format_and_content: gene expression matrix
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| Submission date |
Dec 24, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Magali Soumillon |
| E-mail(s) |
mag.soumillon@gmail.com
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| Organization name |
Harvard University, HSCRB
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| Street address |
Divinity Avenue 7
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02138 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE53638 |
Characterization of differentiating adipose cells by high-throughput single-cell RNA-Seq |
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| Relations |
| BioSample |
SAMN02485705 |
| SRA |
SRX398597 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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