Affymetrix wt terminal labeling kit

Microarray analysis was performed with the GeneChip Mouse Gene 2.0 ST Array (Affymetrix, Santa Clara, CA, USA) with targets derived from total RNA from NAC. Approaches were identical to those recently described (Eisinger et al., 2014 (link)) and included the Ambion GeneChip WT Expression Kit (Ambion, Austin, TX, USA), the Affymetrix WT Terminal Labeling Kit (Affymetrix), and an Affymetrix GC3000 G7 Scanner. Data were extracted and processed in the Affymetrix Command Console v. 3.1.1.1.229 and cDNA synthesis, fragmentation, labeling, array hybridization, staining, and scanning were performed by the Gene Expression Center at the University of Wisconsin-Madison as in previous studies (Eisinger et al., 2013b (link), 2014 (link); Driessen et al., 2014a (link)).

Six samples from each group were randomly selected for the microarray experiment. The total RNA extracted from the MPOA was used with the GeneChip Mouse Gene 1.0 ST array (Affymetrix, Santa Clara, CA). cDNA for array hybridization was synthesized from 200 ng of total RNA using the Ambion GeneChip WT Expression Kit (Ambion, Austin, TX) according to the manufacturer’s specifications. Briefly, double stranded cDNA was synthesized from the total RNA, and then used as a template for the production of single-stranded cRNA synthesis. The cRNA was used as a template for a second round of cDNA synthesis, and the resulting DNA-RNA hybrids were degraded. The cDNA was then fragmented and biotinylated using the Affymetrix WT Terminal Labeling kit (Affymetrix). Labeled cDNA samples were hybridized with the arrays for 16 hours at 45°C, then washed and stained according to the manufacturer’s instructions. Arrays were scanned at 570 nm on an Affymetrix GC3000 G7 Scanner and data was extracted and processed using the Affymetrix Command Console v. 3.1.1.1229. cDNA synthesis, fragmentation, labeling, array hybridization, staining, and scanning were performed by the Gene Expression Center at the University of Wisconsin-Madison.

An ~1 cm portion of the proximal half part of the colon was excised following euthanasia, washed in PBS, and immediately stored in liquid nitrogen. Approximately five animals of the same strain fed the same diet were pooled at equal mass concentration for further RNA extraction. Total RNA was extracted using Direct­zol (Zymo Research) including the DNase digestion step. Then, 100 ng of total RNA was amplified using the Ambion WT Expression Kit from Life Technologies (part number 4411974) and 5500 ng of cDNA was fragmented and labeled using the Affymetrix WT terminal labeling kit (part number 900671) all following the manufacturer’s protocols. Labeled cDNA was hybridized on an Affymetrix Clariom S Assay microarray platform (GPL23038) in ~16 hr of incubation, then washed and stained using an Affymetrix 450 Fluidics Station according to Affymetrix protocols. Finally, arrays were scanned on Affymetrix GSC3000 7G Scanner. Microarray data preprocessing was performed using apt-probeset-summarize from the Array Power Tool (APT) suite (v2.11.3) with the gc-sst-rma-sketch standard method, and the resulting expression values were log-transformed. Microarray probes targeting polymorphic regions in the BXD population were ignored in the process. For probesets targeting a same transcript, only the probeset with the highest value was considered.