GSM6692770: SCH14_scRNA; Homo sapiens; RNA-Seq
Samples processed as single cells were collected at the time of surgical resection and immediately processed. Tumor samples were minced and dissociated using the Human Tumor Dissociation Kit (Miltenyi Biotech, Bergisch Gladbach, Germany) per manufacturer guidelines. The dissociated cell suspensions were then passed through a 40µm filter, pelleted through centrifugation, and resuspended in AutoMACS Rinsing Solution with 0.5% bovine serum albumin (BSA; Miltenyi Biotech). Red blood cell lysis was performed on all samples with Gibco ACK Lysing Buffer and was followed by debris removal via density gradient when necessary (Debris Removal Solution, Miltenyi Biotech). Cell viability was confirmed to be >80% using 0.4% Trypan Blue staining and manual counting with a hemocytometer.   Samples processed as single nuclei were collected at the time of surgical resection and immediately frozen in OCT compound embedding media (Tissue-Tek, Torrance, California) on a pre-chilled aluminum block resting on dry ice, and stored at -80°C. Tissue scrolls were cut at 30 µm using a Cryostat (50-100 scrolls were cut per sample, depending on the tissue size) and maintained at -80°C until the time of nuclei isolation. Lysis buffer (consisting of Tris-HCl, NaCl, MgCl2, Nonidet P40 Substitute, 0.1M DTT, RNase inhibitor, and nuclease free water) was added to the tissue scrolls, which were homogenized using a Pellet Pestle while on ice. Additional lysis buffer was then added, and the mixture was incubated on ice for 5 minutes. The suspension was passed through a 70 µm strainer and centrifuged before being washed with a solution of PBS with 1% BSA and 1U/µl Rnase inhibitor, incubated on ice for 5 minutes, centrifuged, and resuspended in PBS with 1% BSA and 1 U/µl Rnase inhibitor. The nuclei were then labeled with DRAQ5 (Thermo Scientific) and selected using FACS sorting before being carried forward for single nuclei library creation. Single cell and single nuclei suspensions were processed using 10X Chromium Next GEM Single Cell 3' Reagent Kits v3.1 per manufacturer protocols. Briefly, cells/nuclei were added onto the 10X Next GEM Chip G to form Gel Bead-in-Emulsions (GEMs) in the Chromium instrument followed by cell lysis, barcoding, cDNA amplification, fragmentation, adaptor ligation, and sample indexed library amplification.