RNA-Seq of mouse 3 ileum
Ileal tissues were collected from Il22ra1fl/fl;Villin-cre mice after 16 weeks of HFD. RNA was isolated via Trizol extraction methods and then purified for sequencing. Total RNA was quantitated using the Qubit RNA BR assay kit (Thermo Fisher Scientific). RNA quality was determined on an Agilent 4150 Tapestation (G2992AA). Total RNA was treated with 1MBU Baseline-Zero DNase (Epicentre Cat. No DB0711K). RNA was purified and concentrated using 1.8  vol Agencourt RNAClean XP Beads (Beckman Coulter A63987). Concentrations was measured again with Qubit BR RNA assay kit for input into the TruSeq Stranded Total RNA library protocol. Illumina compatible Total RNA cDNA libraries with RiboZero Gold depletion were generated following the TruSeq Stranded Total RNA Reference Guide. Region analysis was performed for each library using Agilent 4150 Tapestation Analysis Software (Version 3.1) with a range of 200600bp to determine average size of each library. Average size and concentration were then used to calculate the molarity of each library. All libraries were pooled and denatured following standard normalization methods. Denatured libraries were loaded onto an Illumina NextSeq 550 v2.5 Mid 150 output reagent cartridge at a final concentration of 1.5pM in HT1 buffer. Illumina Denatured PhiX control library v3 was also included at 1% concentration. Paired end 75bp single index sequencing was performed yielding ~7.5M PE reads per sample.