RNA-seq 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. C57BL/6J mice of both sexes were used for organoid generation. Transcriptomics libraries were constructed using the NEXTflex™ Rapid Directional RNA-Seq Kit (5138-07) using the polyA pull down beads from Illumina TruSeq RNA v2 library construction kit (RS-122-2001) with the NEXTflex™ DNA Barcodes – 48 (514104). The final pool was quantified using a KAPA Library Quant Kit, denatured in NaOH and combined with HT1 plus a 1% PhiX spike. The flow-cell was clustered using HiSeq PE Cluster Kit v3 (Illumina PE-401-3001). Following the clustering procedure, the flow-cell was loaded onto the Illumina HiSeq2000 instrument following the manufacturer's instructions with a 101 cycle paired reads and a 7 cycle index read. The sequencing chemistry used was HiSeq SBS Kit v3 (Illumina FC-401-3001) with 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. The data was sequenced across 3 lanes: 2009_L006, 2009_L007, 2121_L007.