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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jul 15, 2021 |
| Title |
ucMSC-RNA-seq-Old-rep2 |
| Sample type |
SRA |
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| Source name |
mesenchymal stem cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: umbilical cord-derived mesenchymal stem cells (PCS-500-010, lot #63216950)
cell passage/age: PD32
treatment: none
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| Treatment protocol |
human MSC were cultured to PD12 and PD32. Activated and quiescent NSCs (aNSC and qNSC, respectively) were freshly isolated from the subventricular zone of adult (~7 month) and aged (19-21 month) hGFAP-GFP transgenic mice according to established methods (Leeman, D. S. et al. Lysosome activation clears aggregates and enhances quiescent neural stem cell activation during aging. Science (80-. ). 359, 1277–1283 (2018)).
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| Growth protocol |
Human cord blood mesenchymal stem cells (hMSCs) were purchased from ATCC (PCS-500-010, lot #63216949). Cells were grown in low glucose DMEM (Life Technologies 11885-084) supplemented with 10% FBS (Life Technologies 16000-044, lot #1314735) and 1× penicillin/streptomycin (Life Technologies 15140-122) at 37ºC with 5% CO2 and 3% O2. Medium was replaced every 4 days in the absence of passaging. Cells were grown to ~70% confluence and split 1:4 as needed. Cell density was below the concentration needed for reliable detection with a hemocytometer (105 cells/mL), so the growth curve was estimated as 2 population doublings per passage.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from PD12 and PD32 hMSCs using the RNEasy Mini Kit (Qiagen 74104) following the manufacturer’s protocol, including the optional DNase treatment. 100ng of total RNA was depleted of rRNA (NEB, E6310) following the manufacturer’s protocol;NSCs were isolated by FACS using the surface markers prominin-1 (CD133) and the EGF receptor, and GFAP-GFP fluorescence. For sorting, cells were stained with 1:300 EGF-Alexa 647 (Molecular probes, E-35351), and 1:400 Prominin-1-biotin (eBioscience, 13-1331-80). Dead cells were excluded using propidium iodide and cells were sorted on a BD FACS Aria into PBS. aNSCs and qNSCs can be distinguished by the surface expression of EGFR. Using this scheme, aNSC are prominin-1+; GFP+; EGFR+ and qNSC are prominin-1+; GFP+; EGFR-. We performed this experiment in biological triplicate, generating each RNA-seq library from a single animal, using ~400 cells per library.
hMSC rRNA-depleted RNA was used as the template for a stranded RNA-seq library using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, E7760) following the manufacturer’s protocol. mNSC RNA-seq libraries were generated by GENEWIZ LLC. using the SMART-Seq v4 Ultra Low Input RNA Kit for cDNA synthesis and the Nextera XT DNA Library Preparation Kit.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
rRNA-depleted RNA
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| Data processing |
Raw reads were trimmed to remove sequencing adaptors and low quality reads using Trim Galore version 0.4.4 with default parameters.
Reads were aligned to the hg19/mm10 genome assembly using using HISAT2 version 2.1.0 with default parameters.
Transcript-level read count, and subsequent FPKM and TPM measurements, were performed using salmon version 1.0.0
Genome_build: hg19 for human and mm10 for mouse
Supplementary_files_format_and_content: read counts file generated by salmon
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| Submission date |
Mar 02, 2021 |
| Last update date |
Jul 15, 2021 |
| Contact name |
Weiwei Dang |
| E-mail(s) |
weiwei.dang@bcm.edu
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| Organization name |
Baylor College of Medicine
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| Street address |
1 Baylor Plaza
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| City |
Houston |
| State/province |
Texas |
| ZIP/Postal code |
77030 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE156407 |
Age-associated cryptic transcription in mammalian stem cells is linked to permissive chromatin at cryptic promoters (RNA-Seq) |
| GSE156409 |
Age-associated cryptic transcription in mammalian stem cells is linked to permissive chromatin at cryptic promoters |
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| Relations |
| BioSample |
SAMN18106920 |
| SRA |
SRX10206424 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5124499_O_ucMSC_RNA_seq_rep2_salmon.quant.sf.txt.gz |
2.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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