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diff --git a/general/datasets/RTHC_0211_R/experiment-design.rtf b/general/datasets/RTHC_0211_R/experiment-design.rtf new file mode 100644 index 0000000..2b4d4cc --- /dev/null +++ b/general/datasets/RTHC_0211_R/experiment-design.rtf @@ -0,0 +1,7 @@ +<blockquote>
+<p>Parental and BXD lines were received from Jackson Laboratory, or from Oak Ridge Laboratory (BXD43, BXD51, BXD61, BXD62, BXD65, BXD68, BXD69, BXD73, BXD75, BXD87, BXD90), and were bred in the facility of the Neuro-BSIK consortium (VU University Amsterdam). Female mice 3 per strain were housed on sawdust in standard Makrolon type II cages with food (Harlan Teklad 2018) and water ad libitum under specific pathogen free conditions. For the analysis, mice were transferred to the animal facility in Braunschweig and adapted for at least two weeks to the new environment before preparing the spleen cells. All protocols involving mice were approved by national animal welfare committees.</p>
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+<p>For sorting of Tregs and Th cells, splenocytes from 31 BXD recombinant inbred strains as well as from the parental mouse lines DBA/2J and C57BL/6J were isolated by flushing the spleens with erythrocyte-lysis-buffer. Cells were collected by centrifugation, re-suspended in cold FACS-buffer (PBS / 2% FCS / 0,5 mM EDTA). After passing the cells through a 100 µm cell strainer and an additional washing step with FACS-buffer, splenocytes were stained with anti-CD4-APC and anti-CD25-PE for 10 minutes at 4°C, washed and re-suspended in FACS-buffer. CD4+ T cells were separated into CD4+CD25+ Tregs and CD4+CD25- Th cells using a MoFlo cell sorter (Cytomation) and purity of the sorted T cell subsets reached 95-97%.</p>
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+<p>Quality and integrity of the total RNA isolated from 1x10<sup>5</sup> cells was controlled by running all samples on an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies; Waldbronn, Germany). RNA amplification and labeling was done according to manufactures protocol (Small Sample Target Labeling Assay Version II, Affymetrix; Santa Clara, CA). The concentration of biotin-labeled cRNA was determined by UV absorbance. In all cases, 10 µg of each biotinylated cRNA preparation were fragmented and placed in a hybridization cocktail containing four biotinylated hybridization controls (BioB, BioC, BioD, and Cre) as recommended by the manufacturer. Samples were hybridized to an identical lot of Affymetrix MOE430 2.0 for 16 hours at 46°C. After hybridisation the GeneChips were washed and stained using the Affymetrix´s recommended EukGE-WS2v5 protocol for GeneChip® Fluidics FS400 station. Images were scanned using GeneChip® Scanner 3000 under the control of GCOS 1.3 software package (Affymetrix; Santa Clara, CA).</p>
+</blockquote>
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