Multiomic analysis reveals cellular and epigenetic plasticity in intestinal pouches of ulcerative colitis patients [scRNA-seq]

Single cells were loaded onto chip G per the manufacturer’s protocol for the Chromium Single Cell 3’ Library (v3.1) [10X Genomics 1000121].

Section Methods — scRNA library preparation and sequencing, offset —

GEO age characteristics: 66; 63; 36; 41; 50; 70; 66; 63; 36; 41; 50; 70

Section GEO family SOFT, offset —

all individuals presented without clinical symptoms of overt pouchitis

Section Results — Single-cell RNA-seq profiling reveals cellular composition of ileal pouch tissue, offset —

The samples were obtained from a small cross-sectional cohort consisting of six individuals.

Section Discussion, offset —

we performed scRNA-seq (10x Genomics) on paired biopsy samples from the pouch and pre-pouch from 6 UC-IPAA patients

Section Results — Single-cell RNA-seq profiling reveals cellular composition of ileal pouch tissue, offset —

we performed scRNA-seq (10x Genomics) on paired biopsy samples from the pouch and pre-pouch from 6 UC-IPAA patients

Section Results — Single-cell RNA-seq profiling reveals cellular composition of ileal pouch tissue, offset —

SoupX was used with default parameters for the estimation and removal of cell free mRNA contamination for each sample separately

Section Methods — scRNA-seq preprocessing and quality control, offset —

Principal components were corrected using Harmony (v0.1.0) to remove patient-specific effects.

Section Methods — scRNA-seq dimensionality reduction, clustering analysis and visualization, offset —

By combining computational annotation that leveraged previously published data with manual annotation employing well-defined marker genes, we annotated 47 cell types across all lineages

Section Results — Single-cell RNA-seq profiling reveals cellular composition of ileal pouch tissue, offset —

Doublet score was calculated using Scrublet (v0.2.3), and a cut-off score of 0.2 was applied to exclude doublets.

Section Methods — scRNA-seq preprocessing and quality control, offset —

Gene expression for each cell was normalized and log transformed.

Section Methods — scRNA-seq preprocessing and quality control, offset —

Cells were filtered for more than 500 UMIs, fewer than 20,000 UMIs, more than 200 unique genes, fewer than 6,000 unique genes, less than 50% of reads mapping to mitochondrial genes and less than 40% of reads mapping to ribosomal genes

Section Methods — scRNA-seq preprocessing and quality control, offset —

While histopathological evaluation indicated the presence of enteritis in the pouch samples, all individuals presented without clinical symptoms of overt pouchitis

Section Results — Single-cell RNA-seq profiling reveals cellular composition of ileal pouch tissue, offset —

Using paired samples from the same patients allowed us to use the pre-pouch regions as ‘internal control’ for each patient

Section Results — Single-cell RNA-seq profiling reveals cellular composition of ileal pouch tissue, offset —