GSM6401752: CD 3; Homo sapiens; RNA-Seq
De-identified surgical specimens from inflamed terminal ileal mucosa were obtained from CD patients diagnosed by established clinical, radiologic, and histopathologic criteria. Tissues were immediately placed in ice-cold RPMI and processed within 1 h of resection. For comparison, biopsy specimens were obtained from age- and sex-matched healthy controls. To control for variations in immune cell populations residing in the intestinal micro-organization, tissue resections were subjected to sampling using biopsy forceps along the mucosa of the resection specimens, collecting approximately 20 biopsies from each. Biopsies were stored in HypoThermosol® FRS (BioLife Solutions) on ice and later transferred to CryoStor® CS10 (StemCell) in 1.5 ml cryogenic vials. Samples were frozen at -80°C overnight and stored in liquid nitrogen until single-cell suspensions were prepared. Healthy controls consisted of patients evaluated for gastrointestinal symptoms (i.e., abdominal pain, diarrhea, rectal bleeding) who were found to have normal endoscopic evaluations. Whole live cells were washed twice in 1x PBS + 0.04% BSA and immediately submitted to the Core for single cell partitioning. Cells were counted and measured for viability using the Vi-Cell XR Cell Viability Analyzer (Beckman-Coulter, Brea, CA). The barcoded Gel Beads were thawed from -80°C and the cDNA master mix was prepared according to the manufacture's instruction for Chromium Next GEM Single Cell 3' Library and Gel Bead Kit (10x Genomics, Pleasanton, CA).  Based on the desired number of cells to be captured for each sample, a volume of live cells was mixed with the cDNA master mix. A per sample concentration of 500,000 cells per milliliter or better was required for the standard targeted cell recovery of approximately 5,000 cells.  The stock concentration requirements would not change for higher cell recovery numbers. The cell suspension and master mix, thawed Gel Beads and partitioning oil were added to a Chromium Single Cell G chip. The filled chip was loaded into the Chromium Controller, where each sample was processed and the individual cells within the sample were partitioned into uniquely labeled GEMs (Gel Beads-In-Emulsion). The GEMs were collected from the chip and taken to the bench for reverse transcription, GEM dissolution, and cDNA clean-up. The resulting cDNA contained a pool of uniquely barcoded molecules. A portion of the cleaned and measured, pooled cDNA continued to library construction, where standard Illumina sequencing primers and a 10X Genomics unique i7 sample index were added to each cDNA pool. All cDNA pools and resulting libraries were measured using Qubit High Sensitivity assays (Thermo Fisher Scientific, Waltham, MA) and Agilent Bioanalyzer High Sensitivity chips (Agilent, Santa Clara, CA). Libraries were sequenced at between 40,000 and 50,000 fragment reads per cell following Illumina's standard protocol using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit (Illumina, San Diego, CA).  The flow cells were sequenced as 100 X 2 paired end reads on an Illumina HiSeq 4000 HD using HiSeq 3000/4000 sequencing kit and HCS v3.4.0.38 collection software.