title: GSM7869230: Meki,_treated, 2d, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: tumour samples were collected in Splitting Medium (AdDMEM/F12 medium supplemented with HEPES (10 mM), Glutamax, and Pen/Strep) supplemented with 0.1% BSA and RhoKi (10.5 uM). After washing with PBS, specimens were cut in small pieces (1mm3) and incubated for 20 minutes in a tube rotator at 37°C in warm Digestion Medium (PBS 1X, 2mg/mL Dispase I, 1.25mg/mL Collagenase Type II, 100ug/mL DNAse I, and 0,05% FBS) supplemented with RhoKi. The cell suspension was pipetted and incubated on ice to let the larger tissue clumps settle to the bottom of the tube. The surnatant was collected, spun down and the pellet was resuspended in Splitting Medium supplemented with 0.1% BSA and 10 mg/ml Soybean trypsin inhibitor and stored on ice (Fraction 1). Then, the larger undigested clumps were digested again for 10 minutes, and every step previously described was repeated twice until the collection of Fraction 2 and 3. After digestion, Fractions 1, 2, and 3 were combined, filtered (40um nylon cell strainer) and centrifuged. The pellet was then resuspended in ACK lysing buffer supplemented with DNAse I to remove red blood cells from the sample and spun down. Cells were washed with PBS supplemented with 10% FBS and 1x104 cells (concentration 1.000 cells/ul) were submitted for sequencing libraries were generated with GemCode Single Cell 3' Gel Bead and Library Kit v3 (10x Genomics)