|
| Status |
Public on May 07, 2019 |
| Title |
CYP2C19-KO HLCs [AR3098_01] |
| Sample type |
RNA |
| |
|
| Source name |
FCL-iPS, hepatocyte-like cells, iPSC-derived, CYP2C19-KO
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: FCL-iPS
cell type: iPS cell-derived hepatocyte-like cells (iPSC-HLCs)
genotype/variation: CYP2C19-deficient
|
| Treatment protocol |
Hepatic differentiation: Before the initiation of hepatic differentiation, human iPS cells were enzymatically dissociated into clamps by using dispase (Roche), and the clamps were plated onto BD Matrigel Basement Membrane Matrix Growth Factor Reduced (BD Biosciences). These cells were cultured in the MEF-conditioned medium until the cells reach approximately 80% confluence. The differentiation protocol for the induction of definitive endoderm (DE) cells, hepatoblast-like cells, and HLCs was based on our previous reports with some modifications. Briefly, in the DE differentiation, human iPS cells were cultured for 4 days in RPMI1640 medium (Sigma), which contained 100 ng/ml Activin A (R&D Systems), 1×GlutaMAX (Thermo Fisher Scientific), and 1×B27 Supplement Minus Vitamin A (Thermo Fisher Scientific). For the induction of hepatoblast-like cells, the DE cells were cultured for 5 days in RPMI1640 medium (Sigma) which contained 20 ng/ml bone morphogenetic protein (BMP) 4 (R&D Systems), 20 ng/ml FGF4 (R&D Systems), 1×GlutaMAX, and 1×B27 Supplement Minus Vitamin A. To perform the hepatic differentiation, the hepatoblast-like cells were cultured for 5 days in RPMI1640 medium (Sigma), which contained 20 ng/ml hepatocyte growth factor (HGF), 1×GlutaMAX, and 1×B27 Supplement Minus Vitamin A. Finally, the cells were cultured for 11 days in Hepatocyte Culture Medium (HCM, Lonza) without epidermal growth factor (EGF) but with 20 ng/ml oncostatin M (OsM).
|
| Growth protocol |
The two groups of human iPS cells (CYP2C19-deficient iPS cells and wild type iPS cells) were maintained on iMatrix-511 (Nippi)-coated plates with StemFit AK02N medium (Ajinomoto).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using RNeasy Mini Kit.
|
| Label |
Cy3
|
| Label protocol |
Cyanine3 (Cy3) labeled cRNA was prepared from 0.1 μg Total RNA using the Low Input Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop ND-2000 Spectrophotometer.
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| |
|
| Hybridization protocol |
0.6 μg of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 25 μl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer’s instructions. On completion of the fragmentation reaction, 25 μl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to SurePrint G3 Human GE 8x60K Microarray Ver3.0 (Agilent Technologies) 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 Technologies) and 1 minute with 37°C GE Wash buffer 2 (Agilent Technologies).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent SureScan Microarray Scanner (G2600D) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green PMT is set to 100%).
|
| Description |
CYP2C19-KO HLCs
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 12.0.3.1 (Agilent Technologies) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities.
Processed signal intensities were normalized by the global scaling method. A trimmed mean probe intensity was determined by removing 2% of the lower and the higher end of the probe intensities in order to calculate the scaling factor. Normalized signal intensities were then calculated from the target intensity on each array using the scaling factor, so that the trimmed mean target intensity of each array was arbitrarily set to 2500.
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| |
|
| Submission date |
Dec 17, 2018 |
| Last update date |
May 09, 2019 |
| Contact name |
Tomoki Yamashita |
| E-mail(s) |
yamashita-t@phs.osaka-u.ac.jp
|
| Organization name |
Osaka University
|
| Department |
Graduate School of Pharmaceutical Sciences
|
| Lab |
Laboratory of Biochemistry and Molecular Biology
|
| Street address |
1-6 Yamadaoka
|
| City |
Suita |
| State/province |
Osaka |
| ZIP/Postal code |
5650871 |
| Country |
Japan |
| |
|
| Platform ID |
GPL20844 |
| Series (1) |
| GSE123937 |
Gene expression profiles in CYP2C19-deficient human iPS cell-derived hepatocyte-like cells |
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