normalization data transformation protocol
Expression estimates were calculated applying the RMA algorithm in the Bioconductor library 'Oligo' (v1.22.0). ID_REF = VALUE = RMA signal (as log2)
array scanning protocol
Arrays were scanned on an Affymetrix 3000 7G scanner, as described in the Genechip Expression Analysis Technical Manual, section 2 (Eukaryotic Sample and Array Processing), chapter 2 (Eukaryotic Arrays: Washing, Staining and Scanning (P/N 701028, revision 5).
Hybridization of 5.5μg labelled cDNA was done overnight for 17 hours, at 60 rpm, at 45ºC in a Hybridization Oven 640 (Affymetrix). The protocol was conducted as described in the Affymetrix Whole Transcript (WT) Sense Target Labeling Assay Manual, chapter 5 (P/N 701880, revision 5). Washing and staining of the arrays were done on an Affymetrix 450 fluidics station using the protocol FS450_0002, as described in the Affymetrix Whole Transcript (WT) Sense Target Labeling Assay Manual, chapter 5 (P/N 701880, revision 5).
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).
Male Landrace pigs (barrows) were obtained from the Pig Innovation Centre (Sterksel) of Wageningen University. Pigs were individually housed in metabolism pens of 2 m2, equipped with a feeder. Artificial lights were on from 0500 until 1900 and dimmed during the dark period. Pigs had free access to standard pig feed until surgery, and had free access to tap water during the whole study.
sample treatment protocol
At 19 weeks of age pigs underwent surgery for the placement of a cannula in the proximal colon. After 4 to 6 d of postsurgical recovery, pigs were gradually switched to one of two dietary treatments (digestible [control] and resistant starch) that lasted for 14 days. On day 14 after start of the first diet intervention (period 1) biopsies were taken from the proximal colon, after which diets were switched (cross-over). After the second intervention period of 14 days (period 2) biopsies were again taken.
nucleic acid extraction protocol
Total RNA was prepared from biopsies 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).