Dnase digestion

Total RNA was extracted by using TriPure Isolation Reagent (Roche) according to the manufacturer's instructions and subjected to DNase digestion (Roche). First-strand cDNA was obtained from 1 μg of RNA using random hexamer and M-MLV-Reverse Transcriptase (Promega, Italy). Synthesized cDNA corresponding to 25 ng total RNA was used for conventional-(GoTaq DNA Polymerase, Promega) or quantitative-PCR (SYBR Green Master Mix for Light-Cycler 480, Roche), according to manufacturer's instructions. Primers used for RT-qPCR are listed in the Supplementary Table 2.

Approximately 75 mg of the mucosal layer from upper (~6–10 cm distal from the pyloric sphincter, to contain both lower duodenum and upper jejunum) or lower (~25–30 cm distal from pyloric sphincter, mid-jejunum) small intestinal samples was separated from the smooth muscle layer immediately following dissection. Mucosal scrapings were homogenized in lysis buffer (Ambion) using a PowerGen-125 homogenizer (Thermo Fisher Scientific, Toronto, ON) and centrifuged at 12,500 × g for 5 min, and RNA was isolated using the Ambion PureLink RNA Mini Kit per kit guidelines (Thermo Fisher Scientific). RNA was quantified by measuring the absorbance at 260 and 280 nm using Cytation 5 imaging reader (BioTek, Winooski, VT). Four μg of RNA was subjected to DNase digestion (Roche, Mannheim, Germany) at room temperature for 10 min and terminated by the addition of 2.3 mM EDTA and incubating at 70 °C for 15 min. cDNA was generated using the SuperScript Vilo cDNA Synthesis Kit as per the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). qPCR was performed using 100 ng of cDNA, TaqMan Gene Expression master mix, and TaqMan primers for rat 18s or PepT1 (Thermo Fisher Scientific) using a QuantStudio 7 Flex qPCR machine (Applied Biosystems). Relative gene expression was calculated using the ΔΔCt method where each sample was normalized to 18s as the reference gene.