GSM8125137: Perianal5_Sig_nuATAC; Homo sapiens; OTHER
Single Cell Sampling and Isolation  Biopsies for single cell processing were obtained from patients at the time of colonoscopy or upon undergoing abdominoperineal resection (completion proctectomy). At endoscopy, ~4 biopsies per region were collected by the physician for further single cell isolation. From proctectomy specimens, 10-20 biopsies per region were collected by the researcher using an endoscopic forceps prior to pathological grossing. To sample the fistula tract, a cross-section was first excised and then transected to expose the tract lining and facilitate biopsy acquisition. From surgical specimens with patent perianal fistulas, cross sections of the tract were resected with the assistance of the pathologist or pathology PA and preserved in O.C.T. Compound (Sakura) by the Mount Sinai Biorepository Core. FFPE specimens were retrieved from clinical samples previously processed and banked by the Biorepository.   Single Nucleus Isolation  Single cells from biopsies were isolated as described above. Nucleus isolation was carried out if the input cell count was within the range of 100,000 – 1,000,000. Cell lysis and all subsequent steps were carried out on ice or in a centrifuge at 4°C. All centrifugation steps post-lysis were for 5 min at 500 xg rcf. Cells were incubated in 100 L lysis buffer (0.1% NP-40 substitute [Sigma, 11754599001], 0.01% digitonin [MP Bio, 159480], 10% Tween-20 [Sigma, P7949] in wash buffer – 10 mM Tris-HCl pH 7.4 [Sigma, T2194], 10 mM NaCl, 3 mM MgCl2, 0.1% BSA, 1 mM DTT) for 3 min. Immediately upon the completion of lysis, 1 mL chilled wash buffer was added and the sample centrifuged. A total of 2-3 further washes were carried out; final nuclei counts were obtained during wash 2. The final centrifuged nucleus fraction was resuspended in a pre-determined volume of 1X Nucleus Buffer (10X Genomics, cat.no. 2000207) per the manufacturer's guidelines and Nucleus Stock Concentration Table. A final nucleus count was performed to calculate the volume of nucleus stock in 1X buffer to carry forward into the initial Multiome transposition reaction. All nucleus preparation buffers contained 1U/L of RNase Inhibitor (Sigma, 3335399001).  After preparation of the nuclei, the 10X Genomics Single Cell ATAC + Gene Expression kit (PN-1000282) was carried out according to manufacturer's instructions, and a targeted concentration of 10,000 nuclei were loaded per lane of the 10X Chromium Controller. Nuclei were loaded on a Chromium Next GEM Chip J Single Cell Kit (PN-10002834) with GEMs from the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle (PN-1000283) and indexed using the Dual Index Kit TT Set A (PN-1000215) and Single Index Kit N Set A (PN-1000212) according to 10x protocol CG000338 Rev D.