RNA sequencing of Il4ra-deficient small intestinal tuft cells

These are the raw data for an experiment to identify genes regulated by Il4ra signaling in small intestinal tuft cells. Il4ra-fl/fl;Pou2f3-Cre-Ert2/+ mice and littermate controls received tamoxifen chow for 6 days to induce deletion of Il4ra in tuft cells. All mice also received one dose of IL-4 (2µg) + anti-IL-4 (10µg) complexes 8 hours before euthanization and tissue harvest. For small intestinal lamina propria preparations, the first 5 cm of the proximal SI were isolated, Peyer’s patches removed, and the tissue opened longitudinally and cut into 1-2 cm sections. Sections were transferred into 35 mL ice cold HBSS (no Ca+2/Mg+2) containing 10 mM HEPES and shaken vigorously for 30 s. Tissue pieces were filtered through a mesh, stored on ice in RPMI 1640 (Fisher) + 5% FCS then transferred into 15 ml pre-warmed of EDTA [HBSS (no Ca+2/Mg+2) + 10 mM HEPES + 3mM EDTA] and shaken for 10 min at 200 RPM at 37°C. This wash was repeated twice (three washes total) to remove the epithelium, with tissue rinsed between washes with 10ml prewarmed HBSS (no Ca+2/Mg+2) + 10 mM HEPES. Following EDTA washes, tissues were digested in 10 ml prewarmed RPMI 1640 supplemented with 20% fetal calf serum (FCS; Biowest), 1 mg/ml collagenase A (Sigma-Aldrich), 10 mM HEPES, 1 mg/ml DNase I (Sigma-Aldrich) and shaken at 200 rpm at 37°C for 30 min. Digested tissues were vortexed, filtered sequentially through a 100 µm and 40 µm mesh, and washed with ice-cold PBS + 3% FCS. Cells were pelleted by centrifugation (5 min, 1500 rpm), supernatant discarded, and the cell pellet washed and stained for flow cytometry. Single cell epithelial suspensions were generated and stained as described above and then 500 tuft cells (CD45low EpCAM+ CD24+ SigF+) directly into lysis buffer from the SMART-Seq v4 Ultra Low Input RNA Kit (Takara) and cDNA was generated following manufacturer’s instructions. Four biological replicates were collected for each genotype. Each biological replicate represents one mouse. Next-generation sequencing and analysis was performed by the Benaroya Research Institute Genomics Core. Sequencing libraries were generated using the Nextera XT library preparation kit with multiplexing primers, according to manufacturer’s protocol (Illumina), and library quality was assessed using the Tapestation (Agilent). High throughput sequencing was on HiSeq 2500 (Illumina), sequencing dual-indexed and single-end 58 base pair reads. All samples were in the same run with target depth of 5 million reads to reach adequate depth of coverage.