strain: C57BL/6J Sex: male tissue: small intestine, epithelial cell scraping peg plate: G020 labeling batch: 3 individual scraped: researcher3
Treatment protocol
After 3 weeks, at the age of seven weeks, a subset of mice (n=8) were fed a low fat diet (LFD) for 2 weeks. The remaining mice (n=4) remained on the standard laboratory chow (RMH-B) diet. Subsequently, after these 2 weeks, at the age of nine weeks, half of the mice fed the LFD were put on a high fat diet (HFD; n=4), whereas the remaining mice were kept on the LFD (n=4). The chow fed mice remained on the RHM-B diet. After 2 additional weeks, at the age of 11 weeks, all mice were killed, after which the small intestine was removed, cut into 10 equal parts [1=duodenum, 10=terminal ileum], epithelial cells scraped off, snap-frozen in liquid nitrogen, and stored at -80° until analysis. The standard laboratory chow, RMH-B (cat. no. 2181), containing 5% of energy derived from fat, was obtained from ABDiets, Woerden, the Netherlands. The LFD and HFD contained 10% respectively 45% of energy derived from fat (diet formulations based on formulation D12450B resp. D12451; OpenResearch Diets, Research Diets, Inc). Exact diet formulations are available in the supplemental data. Throughout the whole experiment mice had free access to feed and water.
Growth protocol
Four-week old male C57BL/6J mice (N=12) were purchased from Harlan (Horst, The Netherlands) and were housed in pairs in the light- and temperature-controlled animal facility of Wageningen University. Mice had free access to feed and water and prior to the start of the diet intervention they received standard laboratory chow (RMH-B). All animal procedures were conducted under protocols approved by the Local Committee for Care and Use of Laboratory Animals of Wageningen University, the Netherlands.
Extracted molecule
total RNA
Extraction protocol
Total RNA was prepared from scrapings using TRIzol reagent, whereafter purified total RNA was isolated using Qiagen RNEasy columns. RNA integrity was checked on chip analysis (Agilent 2100 bioanalyzer, Agilent Technologies, Amsterdam, the Netherlands) according to the manufacturer's instructions. RNA was judged as suitable for array hybridization only if samples exhibited intact bands corresponding to the 18S and 28S ribosomal RNA subunits, and displayed no chromosomal peaks or RNA degradation products (RNA Integrity Number > 8.0).
Label
biotin
Label protocol
One hundred nanogram of RNA was used for whole transcript cDNA synthesis with the Ambion WT expression kit [catalog number 4411974] (Applied Biosystems/Life Technologies, Nieuwekerk a/d IJssel, The Netherlands).
Hybridization protocol
Hybridization and washing of the Affymetrix GeneChip Mouse Gene 1.1 ST peg arrays were performed on a GeneTitan Instrument (Affymetrix, Santa Clara, CA) according to the manufacturer's recommendations.
Scan protocol
Arrays were scanned on an Affymetrix GeneTitan instrument (Affymetrix, Santa Clara, CA).
Data processing
Expression estimates were calculated applying the RMA algorithm in the Bioconductor library 'Oligo' (v1.28.2).
