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| Status |
Public on Aug 02, 2018 |
| Title |
miRNAs affected in peripheral blood CD34+ cells by stroke |
| Organism |
Homo sapiens |
| Experiment type |
Non-coding RNA profiling by high throughput sequencing
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| Summary |
To understand the differentially expressed miRNAs in the peripheral blood CD34+ cells after stroke (first 24 hours after symptom onset), we have employed mRNA sequencing. Among 45 patients included in the study, 6 patients were chosen depending on the clinical outcome (3 patients with "good" and 3 patients with "bad" clinical outcome after 3 months). Immediately after the collection of the peripheral blood from the patients, mononuclear cells (MNCs) were isolated by density centrifugation. Then, CD34+ cells were isolated from MNCs using MACS columns. The cells were immediately frozen in RNAProtect Cell Reagent. The cell samples were shipped to Seqmatic LLC (Fremont, CA, USA) for RNA isolation and miRNA sequencing. RNA integrity measurements, library preparation and next generation sequencing were conducted at Seqmatic LLC, CA, USA.
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| Overall design |
Around 50-60 mL peripheral blood was collected from stroke patients in the first 24 h after the symptom onset. The blood samples were diluted 2 times with dilution buffer (Ca2+/Mg2+-free PBS with 2mM EDTA) and 35 mL of blood was carefully layered on 15 mL of Lymphoprep (Axis-Shield, Oslo, Norway). After 35 min centrifugation at 400xg, the mononuclear cell (MNC) ring was collected and washed with MACS Buffer (Ca2+/Mg2+-free PBS with 2mM EDTA and 0.5% BSA) 3 times by centrifugation at 300xg. After the last centrifugation, the cells were counted and incubated with CD34 micro beads from Miltenyi according to the manufacturer's instructions. Then, the cells were passed through MACS columns 2 times (1 time through LS and 1 time through MS column) to obtain highly pure CD34+ cell population. The cells were centrifugaed at 300xg for 10 min and the cell pellet was frozen in RNAProtect Cell Reagent from Qiagen immediately. The cells were kept -80 celcius degrees until the shipment to Seqmatic LLC (CA, USA) on dry ice.
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| Contributor(s) |
Aday S |
| Citation(s) |
29237797 |
| Submission date |
Aug 08, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Sezin Aday |
| E-mail(s) |
sezin_aday@yahoo.com
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| Organization name |
University of Pennsylvania
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| Street address |
210 South 33rd Street, Skirkanich Hall
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| City |
Philadelphia |
| State/province |
Pennsylvania |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platforms (1) |
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| Samples (6)
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| Relations |
| BioProject |
PRJNA338121 |
| SRA |
SRP081077 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE85299_RAW.tar |
60.0 Kb |
(http)(custom) |
TAR (of CSV) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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