GSM9225912: D600-JEPI1; Homo sapiens; ATAC-seq
Mononuclear cell suspensions were prepared from dissociated human lung tissue using the detailed protocol "Preparation of Single Cell Suspension from Human Lung Tissue" on protocols.io (dx.doi.org/10.17504/protocols.io.bwr9pd96). After dissection of the epithelium and lamina propria, mononuclear cell suspensions were prepared from dissociated human jejunum tissue using the detailed protocol "Preparation of Single Cell Suspensions of the Intra-Epithelial Layer and Lamina Propria from Human Intestinal Tissue" on protocols.io (dx.doi.org/10.17504/protocols.io.bwq7pdzn). The resulting single-cell suspensions from both tissue sites were cryopreserved and stored in liquid nitrogen. For snATAC-seq, cryopreserved cells were thawed rapidly at 37°C until small ice crystals remained. Cells were transferred into pre-warmed culture medium and gradually diluted to minimize shock. Following centrifugation, pellets were gently resuspended and treated with Benzonase (Millipore Sigma) to reduce cell clumping. Cells were washed in DPBS containing FBS and EDTA, and viability was assessed by fluorescence microscopy-based cell counting (Chemometec NC-3000). Finally, dead cells and contaminating populations (e.g., CD66b+, CD235ab+, and EpCAM+ cells) were removed using magnetic bead–based depletion (Miltenyi Biotec; BioMag Plus Streptavidin). Nuclei were prepared following the 10x Genomics protocol for single-cell ATAC sequencing (CG000169 Rev D) with minor adjustments. Briefly, 1 × 106 cells per sample were pelleted and resuspended in chilled lysis buffer containing detergents (Tween-20, NP-40 substitute, and digitonin) and BSA. After a brief incubation on ice, lysates were diluted in wash buffer and centrifuged to pellet intact nuclei. Nuclei were resuspended in diluted nuclei buffer, counted by hemocytometer, and adjusted to a concentration corresponding to ~8,000 nuclei for loading into the 10x Genomics Chromium Controller for encapsulation and barcoding. Sequencing libraries were prepared using Chromium Next GEM Single Cell ATAC Reagent Kits v2 (10x Genomics) and sequenced on an Illumina NovaSeq X Plus with 2x100 paried-end reads.