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Sample GSM4114014 Query DataSets for GSM4114014
Status Public on May 14, 2020
Title mLN FRC
Sample type RNA
Source name fibroblastic reticulum 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.
Submission date Oct 08, 2019
Last update date May 14, 2020
Contact name Oliver Dittrich-Breiholz
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

Data table header descriptions
VALUE Normalized processed signal intensity values (non logarithmic), averaged across on-chip replicate probes. Measurements of control features were removed.

Data table
A_51_P100021 15
A_51_P100034 1035.53864
A_51_P100052 15
A_51_P100063 239.1835764
A_51_P100084 63.17804347
A_51_P100099 1277.125122
A_51_P100155 1942.180231
A_51_P100174 54.19626246
A_51_P100181 77.72514932
A_51_P100218 15
A_51_P100227 833.2055025
A_51_P100238 15
A_51_P100246 846.0089664
A_51_P100289 705.3143032
A_51_P100298 26.52645902
A_51_P100309 15
A_51_P100327 322.4625372
A_51_P100347 15
A_51_P100379 166.6091439
A_51_P100428 15

Total number of rows: 41174

Table truncated, full table size 865 Kbytes.

Supplementary file Size Download File type/resource
GSM4114014_M3387.txt.gz 9.3 Mb (ftp)(http) TXT
Processed data included within Sample table

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