Ileal Epithelial and Lamina Propria Cell Isolation

Ileal epithelial cells and lamina propria mononuclear cells were isolated from ileal tissue as previously described and stored in RLT buffer (Qiagen). RNA was extracted and purified using RNeasy Plus Mini Kit (Qiagen) and quantified by Nanodrop prior to reverse transcription with iScript cDNA synthesis kit (Bio-Rad). qPCR was performed on an Applied BioSciences Quant Studio 6 Flex Real-time PCR (Applied Biosystems) using PerfeCTa SYBR Green Fast mix, Low ROX (Quanta Biosciences). The thermocycler program was as follows: initial cycle of 95°C for 60 s, followed by 40 PCR cycles at 95°C for 5 s, 60°C for 15 s, 72°C for 15 s. Relative levels of the target genes were determined by calculating the ΔCt to housekeeping gene hprt expression. Primer sequences can be found in Table S3.^{20 (link),69 (link)–72 (link)}