Influx instrument

Isolation of Foxl1+ cells using fluorescent-activated cell sorting was performed using Foxl1–Cre;Rosa–YFP mice. Small intestines were dissected and washed thoroughly with PBS. Intestinal villi were scraped using a coverslip and the remaining tissue was incubated in 30 mmol/L EDTA plus 1.5 mmol/L dithiothreitol and Hank's balanced salt solution on ice for 20 minutes and subsequently incubated in 30 mmol/L EDTA at 37° for 8 minutes to completely remove the epithelium. After vigorous washes, the remaining mesenchymal fraction was collected and cut into small pieces. The mesenchymal tissue was collected by centrifugation and resuspended in 7 mg/mL Dispase II/0.05% trypsin solution (Sigma-Aldrich, St. Louis, MO) at 37° until the solution became cloudy and the mesenchyme was dissociated. A single-cell suspension was obtained by collecting the supernatant and washing with Hank's balanced salt solution before cell sorting using a BD influx instrument (BD Biosciences, San Jose, CA). For RNA isolation, YFP+ cells were lysed and total RNA was isolated by column purification (Agilent Technologies). Messenger RNA (mRNA) was isolated using Poly(A) mRNA isolation magnetic beads and an mRNA sequencing library prepared using the NEBNext RNA library prep kit (New England BioLabs, Inc, Ipswich, MA). RNA sequencing was performed on an Illumina HiSeq instrument.

Intestines from adult Foxm1–hDTR mice were cut open and washed in PBS. Intestinal epithelial cells were separated from mesenchymal cells as described in fluorescent-activated cell sorting and RNA isolation. The crypt epithelium was scraped off and collected. To ensure isolation of pure epithelium, epithelial cells were incubated in 0.05% trypsin to obtain single cells, followed by incubation with an Alexa-488 mouse EpCAM (1:250; Biolegend, San Diego, CA) antibody, and sorted in a BD Influx instrument (BD Biosciences). The remaining mesenchymal tissue was washed to remove any epithelial contamination. From the epithelial and mesenchymal fractions, RNA was isolated and complementary DNA (cDNA) was synthesized. For mesenchymal and epithelial-specific markers as well as hDTR, reverse-transcription PCR was performed using GoTaq (Promega, Madison, WI). The primers used in this experiment are listed in Table 1.

Sort-seq experiments were conducted using a BD Influx instrument. Cells transfected with the biosensor library (CggR-DIP or CggR-180-VST/NNK) and Bxb1 recombinase were sorted to collect GFP⁺, BFP⁻ cells. For second-round FACS, four equal-width gates were set to span the range of log(AFU) covered by the distribution of each library. The ± glucose samples were sorted using the same gates for ∼1.5 h each. Cells for each bin were collected, individually pelleted, resuspended, and plated for expansion. Genomic DNA from 5 M cells was extracted.