Healthy sample
Passage-matched active EoE and normal fibroblasts were processed for scRNA-Seq libraries using 10X Genomics technology. We loaded a targeted total of 5,000 and 10,000 cells into the Chromium X Controller (10x Genomics) on a Chromium Next GEM Chip G (10x Genomics). Libraries were generated using the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 according to the manufacturer's protocol. Quality control was assessed by Qubit Fluorometer (Thermo Fisher Scientific) and 4200  TapeStation System (Agilent). The multiplexed libraries were pooled and sequenced on a NovaSeq 6000 (Illumina) with a S4 flow cell using kit type 200 and a PE10x run configuration for 1 billion total reads. The output was processed with Cell ranger (version 7.1.0) count routine, using the GrCh38 reference genome. Output feature barcoded matrices from cellranger were first applied to scDblFinder for doublet detection with a doublet rate of 5% and standard deviation of 5%. Then we employed Seurat (v5.0)94 to process these matrices, and retained the non-doublet cells having nFeature_RNA > 700, nFeature_RNA < 5000, nCount_RNA > 1000, nCount < 20000, log10GenesPerUMI > 0.8, percent.mt < 10, percent.ribo < 20 and percent.hb < 0.05. After filtering the remaining cells from individual samples, we merged all these samples using the merge() routine of Seurat, performed normalization using the method LogNormalize with a scaling factor of 10000, obtained top 2000 variable features using the function FindVariableFeatures, scaled data (using the routine ScaleData) by regressing with respect to percent.mt and nCount_RNA, and executed PCA with a dimension of 20. Batch correction was performed by Harmony, using the samples as the covariate. A UMAP was generated using the 7 dimensions based on an elbow plot.
NIAID-phs003869
HE157_S2