GSM8125240: Perianal14_Fistula_cytoRNA; Homo sapiens; RNA-Seq
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 cell suspensions were prepared from 4-10 biopsies as previously described21. If cell viability was < 70%, dead cells were depleted using the Dead Cell Removal Kit (Miltenyi, cat.no. 130-090-101). Single cells were loaded onto the 10X Chromium Controller at a targeted concentration of 6000 cells/lane, using the Chromium 3' v3.1 chemistry (PN-1000121), per manufacturer instructions. Cells were loaded on a Chromium Next GEM Chip G Single Cell Kit (PN-1000127) with GEMs from the Chromium Next GEM Single Cell 3' Kit v3.1 (PN-1000268) and indexed using the Dual Index Kit TT Set A (PN-1000215) according to 10x protocol CG000315 Revs B,C.