Crypts were isolated from the small intestine of mice fed with SD or CDAHFD for 3 wk as previously described17 (link). Briefly, the ileum segments were shaken in cold HBSS containing 30 mM EDTA. After vigorous shaking for ~ 300 times in HBSS, the isolated crypts were resuspended in HBSS containing 300 U/mL collagenase (Sigma), 10 µM Y-27632 (Sigma), and 1 mM N-acetylcysteine (Sigma), and shaken at 180 rpm for 5 min at 37 °C on a horizontal shaker (TAITEC). Then, 50 μg/μL Dnase I (Roche) was added, and the sample was mixed by pipetting. Cells were pelleted at 500 g for 5 min at 4 °C and resuspended in washing buffer (DMEM/F12 containing 10 μM Y-27632 and 1 mM N-acetylcysteine), then passed through 40-μm cell strainer (BD Falcon). Paneth cells were stained with Zinpyr-1 (Santa Cruz) and Allophycocyanin (APC) anti-CD24 (clone M1/69,)Abcam) in washing buffer for 10 min at 37 °C. After Paneth cell labeling, the cells were sorted by flow cytometry using a cell sorter (JSAN, Bay Bioscience). Single cells were gated by forward scatter and side scatter. Cells were sorted directly into lysis buffer for RNA isolation (PureLink RNA Mini Kit, Invitrogen). To make a pooled sample, at least 10,000 Paneth cells were sorted from 2 separate mice, and sorting experiment was repeated three times. 2 pools (each pool contains Paneth cells from 6 separate mice) were prepared for each group. Total RNA was isolated using Invitrogen® PureLink RNA Purification System according to manufacturer’s instructions, and cDNA was synthesized using a SMART-Seq. Sequencing libraries were built with the TruSeq RNA Library Prep Kit (Illumina) and then submitted to Illumina NovaSeq 6000 for 100-bp PE reads sequencing. Fragments per kilobase of transcript per million mapped reads (FPKM) values were used, genes with FPKM values below 1 were not included in the analysis, and fold change ≥ 1.5 or ≤ 0.67 was considered differentially expressed.
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