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Status |
Public on May 31, 2023 |
Title |
CD34+ SC_1 (CM245-01) |
Sample type |
RNA |
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Source name |
human umbilical cord blood
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Organism |
Homo sapiens |
Characteristics |
tissue: umbilical cord blood cell type: CD34+ stem cells
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Treatment protocol |
cells were treated on days 1 and 2 with valproic acid (VPA) and 5-azacytidine (5’AZA), respectively. From day 3, the medium was renewed 3 times per week with previous medium containing ascorbic acid and TGF-β1 until day 14.
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Growth protocol |
Human umbilical cord blood (Hu-UCB) samples were obtained from healthy, consenting donors undergoing normal deliveries. The cells were incubated in Iscove’s modified medium (IMDM) supplemented with 12.5% fetal bovine serum, 2.5% horse serum, 2 mM L-glutamine,, 7 μM 1-thioglycerol , 1% penicillin/streptomycin, BMP2, bFGF, VEGF, IGF1), and BMP4
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was extracted from UCB-CD34+- and UCB-CD34+-treated cells and, at different stages of differentiation, cardiac progenitors (CMPCs) and cardiomyocytes (4 pools of quadruplets).
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Label |
Cy3
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Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 50 ng RNA using the One-Color Low Input Quick Amp WT Labeling kit (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >15.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 25× Agilent Fragmentation Buffer and 10x Agilent Gene Expression Blocking Agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent Hi-RPM Hybridization Buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Human GE v2 8x60K Microarray (G4858A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by very slow removing.
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Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
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Description |
Gene expression in CD34+ stem cells from blood cord
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Data processing |
The scanned images were analyzed with Feature Extraction Software 11.5 (Agilent) using default parameters (protocol GE1_1105_Oct12 and Grid: 039494_D_F_20150612) to obtain background subtracted and spatially detrended Processed Signal intensities. The data were filtered and normalized using Agilent Genespring GX software
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Submission date |
Dec 23, 2022 |
Last update date |
May 31, 2023 |
Contact name |
Anne ARIES |
E-mail(s) |
ariesa@ghrmsa.fr
|
Organization name |
IRHT
|
Street address |
87 Av d'Alkirch
|
City |
Mulhouse |
ZIP/Postal code |
68100 |
Country |
France |
|
|
Platform ID |
GPL17077 |
Series (1) |
GSE221650 |
Deciphering the cardiovascular potential of human CD34+ stem cells |
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