|
| Status |
Public on May 14, 2020 |
| Title |
mLN LEC |
| Sample type |
RNA |
| |
|
| Source name |
lymphatic endothelial cells isolated from mesenteric lymph node (gp38+ CD31+)
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6NCrl
|
| Treatment protocol |
For isolation of CD45- stem cells, the mesenteric lymph node (mLN) of wildtyp mice were removed, and LNs were digested at 37° for 30 min with 1 mg/ml Collagenase 8 in RPMI 1640/10% FCS. CD45- cells were isolated using the MACS technique following the instructions provided by Miltenyi. Stromal cell subsets were sorted using fluorochrome-coupled antibodies (CD45- CD24- CD31 gp38). The mean purity of stromal cell subsets was 88% ± 1.5. The SCs were used for mRNA isolation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Sorted cells from mLN were isolated and total RNA was isolated according to the manufacturer´s protocol (RNeasy Micro Kit, Qiagen, Hilden, Germany).
|
| Label |
Alexa555
|
| Label protocol |
30-50ng of total RNA were used to prepare aminoallyl-UTP-modified (aaUTP) cRNA (Amino Allyl MessageAmp™ II Kit; #AM1753; Thermo Fisher Scientific) applying one-round of amplification as directed by the company, except for a twofold downscaling of all reaction volumes. The labeling of aaUTP-cRNA was performed by use of Alexa Fluor 555 Reactive Dye (#A32756; Thermo Fisher Scientific) as recommended in the manual of the Amino Allyl MessageAmp™ II Kit (twofold downscaled reaction volumes).
|
| |
|
| Hybridization protocol |
cRNA fragmentation, hybridisation and washing steps were carried-out as recommended in the ‘One-Color Microarray-Based Gene Expression Analysis Protocol V5.7’, except that 400ng of each fluorescently labelled cRNA population was used for hybridization.
|
| Scan protocol |
Slides were scanned using the Agilent Micro Array Scanner G2565CA (pixel resolution 5 µm, bit depth 20).
|
| Data processing |
Data extraction and normalization were performed with the “Feature Extraction Software V10.7.3.1” by using the recommended default extraction protocol file: GE1_107_Sep09.xml. Measurements of on-chip replicates were averaged using the geometric mean of processed intensity values of the green channel, ‘gProcessedSignal’ (gPS) to retrieve one resulting value per unique non-control probe. Single Features were excluded from averaging, if they i) were manually flagged, ii) were identified as Outliers by the Feature Extraction Software, iii) lay outside the interval of ‘1.42 x interquartile range‘ regarding the normalized gPS distribution of the respective on-chip replicate population, or, iv) showed a coefficient of variation of pixel intensities per Feature that exceeded 0.5. Averaged gPS values were normalized by Quantile normalization aproach. Finally, a lower intensity threshold (surrogate value) was defined based on intensity distribution of negative control features. This value was fixed at 15 normalized gPS units. All of those measurements that fell below this intensity cutoff were substituted by the respective surrogate value of 15.
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| |
|
| Submission date |
Oct 08, 2019 |
| Last update date |
May 14, 2020 |
| Contact name |
Oliver Dittrich-Breiholz |
| E-mail(s) |
dittrich.oliver@mh-hannover.de
|
| Organization name |
Medical School Hannover
|
| Department |
Research Core Unit Genomics
|
| Street address |
Carl-Neuberg-Str. 1
|
| City |
Hannover |
| ZIP/Postal code |
30625 |
| Country |
Germany |
| |
|
| Platform ID |
GPL7202 |
| Series (2) |
| GSE138594 |
Dietary lipids accumulate in macrophages and stromal cells and change the microarchitecture of the mesenteric lymph node II |
| GSE138595 |
Dietary lipids accumulate in macrophages and stromal cells and change the microarchitecture of the mesenteric lymph node |
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