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Introduction Barrett’s oesophagus (BE) is the metaplastic transition of squamous oesophageal epithelium to a columnar, intestinal mucosa. Although epigenetic reprogramming is known to accompany this transition, the cell-type–specific DNA methylation changes underlying the loss of squamous identity and gain of intestinal phenotype have not previously been characterised in purified human cell populations.Methods We performed differential methylation analysis on FACS-sorted squamous, columnar and immune cell fractions from healthy controls and BE patients collected using the capsule sponge device (n=15). Cell-type composition of sorted cell fractions was independently assessed using reference-based deconvolution of whole-genome methylation profiles. Differentially methylated regions (DMRs) were intersected with a multi-tissue methylation atlas1 to assign each DMR to oesophageal, gastric, small-intestinal, or immune cell lineages. For each atlas-assigned region, we computed Δβ (Healthy − BE) and compared the distributions across annotated cell lineages.Results Across 14,700 atlas-annotated DMRs (4,557 squamous, 6,427 immune, 3,746 columnar), lineage-specific patterns were evident. Squamous-associated DMRs were strongly enriched for oesophageal epithelial regions (n=289) and showed a negative Δβ shift, indicating higher methylation in BE and supporting silencing of squamous regulatory elements. Columnar DMRs were enriched for small-intestinal (n=24) epithelial annotations and showed a broadly positive Δβ shift (Healthy – BE), consistent with hypomethylation in BE at gastric- and intestinal-annotated loci. This pattern suggests increased gastric and small-intestinal epithelial fractions in BE samples relative to controls. In particular, small-intestinal–annotated DMRs showed hypomethylation, consistent with a shift towards an intestinal-associated phenotype in the BE samples. Immune cell DMRs were numerous, with granulocytes (n=40) and B cells (n=31) contributing most to the epigenetic landscape of the BE microenvironment.Conclusions Purified BE cell populations exhibit a cell-type specific methylation shift characterized by hypomethylation of small-intestinal epithelial regions. These enriched columnar cell-fraction DMRs could serve as novel biomarkers to detect BE from capsule sponge, and warrant further validation in a larger clinical cohort.Abstract O73 Figure 1A estimated cell-type proportions for each sample based on deconvolution using a multi-tissue reference atlas. B Distribution of average beta methylation values from reference atlas’ top 1000 methylation sites per sorted cell type. C Magnitude and direction of DMRs comparing squamous and gastric cell fractions between healthy and BE