Small RNA transcriptomics comparison of mouse small intestinal organoids grown to be normally differentiated, enriched for Paneth cells or enriched for goblet and enteroendocrine cells.

Murine enteroids were generated as described previously (Sato et al., 2009; Sato and Clevers, 2013; Yin et al., 2014). Briefly, mouse small intestinal crypt pellets were suspended in 200μl phenol-red free Matrigel (Corning), seeded in small domes in 24-well plates. Enteroid media containing EGF, Noggin and R-spondin (ENR; (Sato et al., 2009)) was then overlaid. On d2, d5 and d7 post-crypt isolation, ENR media was changed to include additional factors for each cell-type specific condition: 3μM CHIR99021 (Tocris) and 10μM DAPT (Tocris) [Paneth cells]; 2μM IWP-2 (Tocris) and 10μM DAPT [goblet and enteroendocrine cells] (Yin et al., 2014). On day eight post‑crypt isolation, organoids were extracted from Matrigel using Cell Recovery Solution (BD Bioscience), rinsed in PBS and RNA was extracted using miRCURY RNA Isolation Tissue Kit (Exiqon) according to the manufacturer’s protocol. Enteroids were generated from three separate animals for each condition, generating three biological replicates. Enteroids were generated from three separate animals for each condition, generating three biological replicates. C57BL/6J mice of both sexes were used for organoid generation. Transcriptomics libraries were constructing using the TruSeq Small RNA Library Prep Kits (15004197 Rev.G). The final pool was quantified using a KAPA Library Quant Kit, denatured in NaOH and combined with HT1 plus a PhiX spike. The libraries were hybridized to the flow-cell using TruSeq Rapid Duo cBot Sample Loading Kit (Illumina CT-403-2001) and the flow-cell was loaded onto the Illumina HiSeq2500 instrument following the manufacturer’s instructions with a 51 cycle single read and a 7 cycle index read. The sequencing chemistry used was HiSeq SBS Rapid Kit v2 (Illumina FC-402-4022) with a single read cluster kit (Illumina GD-402-4002), HiSeq Control Software 2.2.68 and RTA 1.18.66.3. Reads in bcl format were demultiplexed based on the 6bp Illumina index by CASAVA 1.8, allowing for a one base-pair mismatch per library, and converted to FASTQ format by bcl2fastq.