|
| Status |
Public on Jul 17, 2016 |
| Title |
CX3CR1high cells vs. Ly6c+ CD11b+ monocytes |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Pooled CX3CR1high CD11b+ CD11c+ cells derived from CX3CR1gfp/+ mice
|
| Organism |
Mus musculus |
| Characteristics |
strain/background: CX3CR1gfp/+ (PMID: 22403066)
tissue source: intestinal lamina propria
cell type: CX3CR1high CD11b+ CD11c+ cells
sample type: pooled
|
| Treatment protocol |
Murine large intestinal lamina propria cells were isolated from CX3CR1gfp/+ mice as previously described (PMID: 22403066).
|
| Growth protocol |
None
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cells using an miRNeasy Mini kit according to the manufacturer’s instructions (Qiagen). The quality of the RNAs was estimated by using the RNA 6000 Nano LabChip Kit on the Agilent 2100 Bioanalyzer (Agilent).
|
| Label |
Cy5
|
| Label protocol |
25 ng of total RNA was labeled using low RNA Input Linear Amp kit (Agilent). In brief, cDNA was reverse transcribed from 25 ng of total RNA with an oligo-dT-T7 promoter primer and MMLV-RT. Fluorescent antisense cRNA was synthesized with cyanine 5-CTP (Cy5-CTP) or 3-CTP (Cy3-CTP), and T7 polymerase. The fluorescent-labeled antisense cRNA was purified with RNeasy columns (Qiagen) according to manufacturer's protocols and resuspended in water. The purified products were quantified at A650nm for Cy5-CTP and at A552nm for Cy3-CTP with Agilent 2100 Bioanalyzer (Agilent).
|
| |
|
| Channel 2 |
| Source name |
Pooled blood Ly6c+ CD11b+ monocytes derived from C57BL/6J mice
|
| Organism |
Mus musculus |
| Characteristics |
strain/background: C57BL/6J
tissue source: blood
cell type: Ly6c+ CD11b+ monocytes
sample type: pooled
|
| Treatment protocol |
Murine large intestinal lamina propria cells were isolated from CX3CR1gfp/+ mice as previously described (PMID: 22403066).
|
| Growth protocol |
None
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cells using an miRNeasy Mini kit according to the manufacturer’s instructions (Qiagen). The quality of the RNAs was estimated by using the RNA 6000 Nano LabChip Kit on the Agilent 2100 Bioanalyzer (Agilent).
|
| Label |
Cy3
|
| Label protocol |
25 ng of total RNA was labeled using low RNA Input Linear Amp kit (Agilent). In brief, cDNA was reverse transcribed from 25 ng of total RNA with an oligo-dT-T7 promoter primer and MMLV-RT. Fluorescent antisense cRNA was synthesized with cyanine 5-CTP (Cy5-CTP) or 3-CTP (Cy3-CTP), and T7 polymerase. The fluorescent-labeled antisense cRNA was purified with RNeasy columns (Qiagen) according to manufacturer's protocols and resuspended in water. The purified products were quantified at A650nm for Cy5-CTP and at A552nm for Cy3-CTP with Agilent 2100 Bioanalyzer (Agilent).
|
| |
|
| |
| Hybridization protocol |
Before hybridization, 825ng labeled cRNA of each product was fragmented and mixed with control targets and hybridization buffer according to the manufacturer's protocol (Agilent). Hybridizations were done for approximately 17h at 65°C. The slides were washed according to the manufacturer's manual.
|
| Scan protocol |
The array was scanned with 5µm resolution using an Agilent G2565AA DNA microarray scanner. Images were quantified using Agilent Feature Extraction Software (version 10.5.1.1).
|
| Description |
Cy3-blood vs Cy5-colon
|
| Data processing |
Agilent Feature Extraction Software (version 10.5.1.1) was used for background subtraction, LOWESS normalization, and dye-normalization. Data were analyzed using the bioinformatics software GeneSpring 13.0 (Agilent).
|
| |
|
| Submission date |
Mar 25, 2016 |
| Last update date |
Jul 17, 2016 |
| Contact name |
Daisuke Okuzaki |
| E-mail(s) |
dokuzaki@biken.osaka-u.ac.jp
|
| Phone |
+81-6-6879-4935
|
| Organization name |
Osaka univ.
|
| Department |
Immunology Frontier Research Center
|
| Lab |
Human Immunology （Single Cell Genomics)
|
| Street address |
Yamadaoka 3-1
|
| City |
Suita |
| State/province |
Osaka |
| ZIP/Postal code |
565-0871 |
| Country |
Japan |
| |
|
| Platform ID |
GPL10333 |
| Series (2) |
| GSE79628 |
Microarray analysis of CX3CR1high CD11b+ CD11c+ cells in murine intestinal lamina propria |
| GSE79629 |
Microarray analysis of CD14+ CD163high CD160high cells in human intestinal lamina propria and CX3CR1high CD11b+ CD11c+ cells in murine intestinal lamina propria |
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