Two tissue biopsy specimens were obtained from endoscopically and histologically normal prepouch ileum of every eligible subject. The samples were then immediately suspended in RNAlater (Qiagen, Venlo, Netherlands) stabilizing reagent to deter RNA degradation and stored at −80°C. Total RNA was extracted with the miRNeasy Mini Kit (Qiagen) in 2 batches. NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA) and Bioanalyzer 2100 (Agilent, Santa Clara, CA) were used to determine RNA concentration, quality, and purity. Only samples with an RNA integrity number ≥5.0 were considered for further analysis.27 Additional information on RNA integrity number cutoffs is provided in Supplementary Materials and Methods.
