GSM9030790: 001_BX_COM; Homo sapiens; RNA-Seq
Tumor tissue specimens of six OS patients were collected at Connecticut Children's Medical Center (CCMC) or Children's Hospital Colorado (CHCO). All specimen collection and experiments were reviewed and approved by the Institutional Review Board of CCMC or CHCO, respectively. Written informed consent was obtained prior to acquisition of tissue from patients. Diagnosis was confirmed by pathologist assessment. CCMC-sourced specimens were stored in MACS tissue storage solution (Miltenyi Biotec, Bergisch Gladbach, Germany), then transferred to The Jackson Laboratory for Genomic Medicine for the tissue dissociation process. For CCMC samples, the tissue specimens were minced and enzymatically dissociated in DMEM medium supplemented with Collagenase II (250U/ml, Gibco #17101-015, Waltham, Massachusetts, USA) and DNase (1μg/ml, Stemcell #07900, Cambridge, Massachusetts, USA) for up to 3 cycles of 37 °C 15 minutes dissociation with agitation. After each cycle, undigested tissue pieces were settled down by gravity and supernatant was transferred to cold Buffer I solution (10% FBS/DMEM medium supplemented by EDTA (2mM) and 2% BSA (Lampire #7500854, Pipersville, Pennsylvania, USA). Fresh dissociation solution was applied to the undigested tissue for next digestion cycle. Cells collected from each cycle were merged, spun down and resuspended with ACK lysis buffer (Gibco #A1049201) to remove red blood cells. After 3 minutes on ice, lysis reaction was quenched by adding Buffer I solution. After centrifugation, cells were resuspended in Buffer I solution and strained through a 70-μm cell strainer. Cells were stained with propidium iodide (PI) and Calcein Violet (Thermo Fisehr Scientific). PI-negative and Calcien Violet-positive viable cells were sorted out using FACSAria Fusion system (BD Biosciences, Franklin Lakes, New Jersey, USA). For CHCO samples, tissue specimens were viably cryopreserved in 90% FBS 10% DMSO immediately after collection. Tissue specimens were thawed at 37 °C, then enzymatically dissociated in a 0.1% DNase and Liberase 400g/mL (Roche/Sigma-Aldrich, St. Louis, Missouri, USA) cocktail. Cells were then selected for viability using the FACSAria I cell sorter (BD Biosciences) in the Allergy and Clinical Immunology Flow Cytometry Facility at the Division of Allergy and Clinical Immunology, University of Colorado School of Medicine. Cells were washed and resuspended in PBS containing 0.04% BSA. Cells were counted on Countess II automated cell counter (Thermo Fisher, Waltham, Massachusetts, USA), and up to 12,000 cells were loaded per lane on 10x Chromium microfluidic chips (10x Genomics, Pleasanton, California, USA). Single-cell capture, barcoding, and library preparation were performed using the 10x Chromium X version 3 chemistry, and according to the manufacturer's protocol. cDNA and libraries were checked for quality on Agilent 4200 Tapestation and quantified by KAPA qPCR before sequencing using a Novaseq 6000 (Illumina, San Diego, California, USA) v1.5 cycle flow cell lane at 100,000 reads per cell, with a 28-10-10-90 asymmetric read configuration.