GSM8178750: Venetoclax, JE025, scRNASeq; Homo sapiens; RNA-Seq
After tumor isolation, fat, fibrous and necrotic areas were removed from the tumor sample in a tissue culture hood (within 2 minutes to avoid cell viability loss). Then, tumor was cut into 1-2 mm pieces and transferred to a gentleMACS C Tube containing 4.7 mL of sterile media (Knockout DMEM/F12), 200 μl of Enzyme H, 100 μl of Enzyme R, and 25 μl of Enzyme A). C Tubes were placed in the gentleMACS Dissociator under the “Medium” Tumor type (gentleMACS Program 37C_h_TDK_2) for 18 minutes. Then, samples were filtered through a 70μm strainer (previously primed with 2 ml of sterile media). Samples were centrifuged ​​at 250 x g at 4°C for 8 minutes. Then, resuspended in 400 μl of red blood cell lysis buffer and incubated on ice. After 2 minutes, at least 3 times the volume of RBC was added to rebalance osmolarity and the sample was centrifuged at 250 x g at 4°C for 5 minutes and resuspended in HBBS + 0.04% BSA. Samples were then examined for cell viability and counts using the Countess II Dissociated cells were processed for single-cell gene expression capture (scRNA-seq) using the 10X Chromium 3' Library and Gel Bead Kit (10x Genomics), following the manufacturer's user guide. After Gel bead-in-EMulsion Reverse Transcription (GEM-RT) reaction, 12-15 cycles of polymerase chain reaction (PCR) amplification were performed to obtain cDNAs used for RNA seq library generation Libraries were prepared following the manufacturer's user guide and sequenced on the Illumina NovaSeq 6000 Sequencing System at the Columbia University Single Cell Analysis Core