We compared gene expression profiles of RNAs from EGFP-positive and negative fibroblasts from the colon of Il11-Egfp reporter mice (n = 3 mice) on day 7 after DSS administration. To isolate EGFP+ and EGFP- cells by a cell sorter, colon tissues were minced with scissors and digested in RPMI1640 containing 0.25 mg/mL Liberase (Sigma), 50 μg/mL DNase (Roche), 100 U/mL Penicillin, 100 μg/mL Streptomycin, and 5% (v/v) fetal bovine serum (Gibco) for 60 min. EGFP+ and EGFP- cells among Ter119-CD45- CD31- EpCAM- cell populations were enriched using MojoSort Mouse anti-APC Nanobeads (BioLegend) and MojoSort Magnet (BioLegend) and sorted by BD FACSAriaTM III Cell Sorter (BD Biosciences). Total RNAs were subjected to RNA-seq analysis. We collected roughly 10,000 fibroblasts per each group, and purity of EGFP+ fibroblasts was ~90%. RNA-seq analyses were performed using the 5′Tag-seq method as described previously43. Briefly, polyA RNAs were trapped by oligo-dT-immobilized magnetic beads and reverse-transcribed by using Superscript IV (Thermo Fisher Scientific). Then, polyC tailing and 2nd strand synthesis were performed, and cDNA was amplified by PCR. Resulted cDNA products were converted to Ion Torrent sequencing libraries using NEBNext UltraII FS library prep kit (New England Biolabs). RNA sequencing was performed with Ion 540 Kit-Chef, Ion 540 Chip Kit, and Ion Genestudio S5 Sequencer (Thermo Fisher Scientific) according to the manufacturer’s instructions. Adapter trimming and quality filtering of resulting fastq files were performed by using Cutadapt-v1.1844. Trimmed reads were mapped to the Refseq mm10 RNA using Bowtie2-2.2.545 with the following parameters: t -p 16 –N 1 -D 200 –R 20 -L 20 -i S,1,0.50 -nofw. Tag numbers of each gene represented the expression level of each gene and were used as count data. Genes with tag number ≥10 were deemed low expressed and were excluded from the analysis. The sample to sample normalization was performed with R (version 3.5.1) and TCC package46. Normalized data were then tested for differential gene expression using the TCC package, which integrates the edgeR package47. Genes with adjusted P < 0.05, fold-change >2 were identified as statistically significant differentially expressed genes. GO enrichment analysis of differentially expressed genes were performed using the DAVID Bioinformatics Resources (https://david.ncifcrf.gov/home.jsp). Data were deposited in NCBI as a GEO accession number GSE141644 and GSE164232.

To identify target genes by IL-11 in the colon, we intravenously injected 10 μg of IL-11R agonist (provided by K. Tsumoto) into wild-type mice (n = 3 mice). IL-11R agonist is a modified version of human IL-11, where the mutations were introduced into the linker region of human IL-11 to increase stability and biological activities to 10 fold higher than the original IL-11. The detailed characterization was described elsewhere48. Mice were sacrificed at 3 h after injection. Total RNAs were extracted from the colon of mice by using Sepasol II Super. Labeled cRNAs were prepared from total RNA using the Agilent’s Quick Amp Labeling Kit (Agilent). Following fragmentation, cRNA were hybridized to SurePrint G3 Mouse Gene Expression 8x60K (Agilent) according to the manufacturer’s instruction. Raw data were extracted using the software provided by Agilent Feature Extraction Software (v11.0.1.1). The raw data for the same gene were then summarized automatically in Agilent feature extraction protocol to generate raw data text file, providing expression data for each gene probed on the array. Array probes that have Flag A in samples were filtered out. The selected processed signal value was transformed by logarithm and normalized by quantile method. Statistical significance of the expression data was determined using fold change and local-pooled-error test49 in which the null hypothesis was that no difference exists among two groups. Hierarchical cluster analysis was performed using complete linkage and Euclidean distance as a measure of similarity. Gene-Enrichment and Functional Annotation analysis for significant probe list was performed using Gene ontology (http://geneontology.org). All data analysis and visualization of differentially expressed genes was conducted using R 3.0.2 (www.r-project.org). Data were deposited in NCBI as a GEO accession number GSE141643 and GSE141644.

For comparing enriched genes in IL-11+ fibroblasts and IAFs, we used Gene Set Enrichment Analysis (GSEA) software (version 4.1, Broad Institute) and p-value was calculated by two-tailed Kolmogorov-Smirnov test50,51.

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