GSM5155199: Patient 5 tissue of an osteosarcoma; Homo sapiens; RNA-Seq
After removing the fat, visible vessels and surrounding necrotic area, a small fragment tumor tissues were cut and rinsed with clod Dulbecco's phosphate-buffered saline (DPBS, Gibco, C14190500BT). Then, we cut the tissues into approximately 1 mm3 pieces on ice and transferred into DPBC and washed twice. We used collagenase 2 to digest the tissues into single cells for 45 min at 37℃. After filtered by 100 μm cell strainer in the DPBS with 1% fetal bovine serum (FBS) (Gibco, 10091148), the suspended cells were centrifuged at 300 g for 5 min. After discarding the supernatant, we removed red blood cells using 1×RBC lysis buffer (10× diluted to 1×, BioLegend, B250015) for 5 min and passed the cells through a 40μm cell strainer. The final single cell suspension were re-suspended in DPBS with 1% FBS after washing twice with DPBS. Viability was confirmed to be > 80% in all samples via trypan blue (0.4%, Gibco, 420301) and the cell suspensions were kept on ice to prepare for 10× Genomics. The final single cells from each sample were loaded on a 10× Genomics Chromium Single-Cell Chip along with the single cell master mix and single cell 3' gel beads (10× Genomics) to generate single-cell gel bead-in-emulsions (GEMs). According to the manufacturer's instructions the samples were processed by 10× Genomics V3 barcoding chemistry kits. mRNA in drops were undergo the reverse transcription reactions and then the cDNA amplification was followed by the instructions. And the single cell libraries were sequenced on an Illumina HiSeq X Ten instrument.