Single cell RNA-seq of ileal biopsies from IBD patients
Crohn's disease arises through host-environment interaction, with abnormal gene expression resulting from disturbed pathway activation or response to bacteria. Single cell RNA-sequencing of ileal tissue from 2 paediatric Crohn's disease patients was performed, identifying populations of CD8+ effector memory T cells (CD8+ Tem), memory B-cells, monocytes, epithelial cells and plasma cells within the ileal tissue. Specialised epithelial cells driving differential expression of S100A8 and S100A9 and associated with defence to bacterium were identified, as well as IL17-signalling associated pathways in monocyte and epithelial cell populations. Overall design: IBD patient ileal biopsies were collected and digested with liberase TM (90 minutes, +37C), within 30 minutes from biopsy. Single cell suspensions of whole ileal tissue was then processed through the Drop-seq in which cells were co-encapsulated with genetically barcoded beads. 1000 STAMPS (beads exposed to a single cell) for each biopsy were taken further for library preparation. Prepared libraries were run on an Illumina NextSeq (1 × 105 reads/cell). Read filtering, barcode and UMI counting were performed using bcl2fasq software from Illumina and alignment to the Hg_19 human reference genome was performed using STAR. Data analyses were run using the python-based Scanpy with R (3.6.0). Genes (detected <10 cells) and cells (20% or more counts contributed by mitochondrial genes) were filtered. Data was normalised using Scran. A single-cell neighbourhood graph, with data integrated from separate tissue samples, was computed using BBKNN. Data were visualised using Uniform Manifold Approximation and Projection (UMAP), with Leiden algorithm cell clustering. Cell type annotation was performed using SingleR (database: BlueprintEncodeData) and enrichment analysis (ToppGene and EnRICHR) using the top 50 cluster marker genes.