GSM4146147: 4B; Mus musculus; RNA-Seq
A pool of 6 pieces of the tumour from different mice were used for each condition. Pieces of tumour were transferred into gentleMACS C tubes (Miltenyi Biotec) containing dissociation solution composed of: 5 ml of RPMI media, Protease from Bacillus licheniformis (5 mg/ml), CaCl2 (10 mM) and according to the given condition, Actinomycin D (2nM). Tubes were mixed for 1 hour using the gentleMACS dissociator either at 4°C or gentleMACS OctoDissociater with heaters set to 37°C. Immediately after dissociation, all tubes were kept on ice while all solutions were pre-chilled in the ice. Cell suspension was filtered through a 30 µm strainer. Cells were spun down (5 min, 800 g). The medium was discarded and the cells were dissociated in 15 mL of ice cold ACK solution (0.15 M NH4Cl; 10 mM KHCO3; 0.1 mM EDTA; pH 7.3) to remove erythrocytes. The cells suspension were immediately spun (5 min, 800 g). The debris were removed using debris removal solution (Miltenyi Biotec) according to the manufacturer's protocol. Next, the Dead Cells Removal Kit (Miltenyi Biotec) was used to remove dead cells. The collected effluent was spun (5 min, 800 g), supernatant removed and the cells were resuspended to final concentration of 2500 cell/μl in PBS-/-. ScRNA-Seq libraries were prepared using the SureCell WTA 3' Library Prep Kit for the ddSEQ System (Illumina/Bio-Rad) according to the manufacturer´s protocol.
GEO Accession
GSM4146147