RNA sequencing (RNA-seq) was carried out at the sequencing core at the Fox Chase Cancer Center. Transcriptome analysis was performed on RNA isolated from fresh sorted LinSca1+c-kithiCD135 cells from healthy mice or mice experiencing GVHD. Total RNA was isolated from cells using an RNeasy Mini Kit (QIAGEN) and RNA-seq libraries were prepared using SureSelect RNA Library Preparation kits (Agilent Technologies). Samples were run on a HiSeq 2000 sequencing system (Illumina), and at least 37.5 × 106 single-end reads were obtained per sample. Expression was evaluated by determining the fragment per kilobase per million reads values. Using 1-way ANOVA, we selected transcripts with P < 0.01 and q < 0.01 for comparing paired groups and with at least a 1.5-fold difference from the means for the paired groups. Raw sequence reads were aligned to the mouse genome (mm10) using the Tophat algorithm; the Cufflinks algorithm was implemented to assemble transcripts and estimate their abundance. Cuffdiff was used to statistically assess expression changes in quantified genes in different conditions. FDR < 5% and FC ≥ 2 were used as cutoffs to identify significantly changed genes, as described previously (70). RNA-seq data were deposited in the NCBI’s Gene Expression Omnibus database with accession number GSE143788.

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