GSM9157705: Ctrl-D; Mus musculus; RNA-Seq
Tumors were collected from the Ctrl-D and Eed iKO-D mice and cut into small pieces (< 1mm3) with razor blades in 10cm plate. Chopped tumors were incubated with 0.25% collagenase (Millipore Sigma) in HBSS for 45 minutes. After collagenase incubation, chopped tumors were treated with 1 unit DNase (Millipore Sigma) for 10 minutes. Collect the tumors and medium filtered through 100μm filters and transfer the tumors from filters into 10cm plate. Tumors were incubated 0.25% Trypsin/EDTA (Corning) at 37C for 30 minutes. Dissociated tumor single-cell suspensions were sequentially strained through 100μm and 40μm filters. Viable cells were isolated by Dead Cell Removal kit (Miltenyi Biotec) according to the manufacturer's instructions. Library preparation was performed with the Single Cell 10X Chromium 3'v2 kit (10X Genomics) following the manufacturer's protocol. Target number of captured cells ranged from 8013-19511 cells. Sequencing was performed on a HiSeq 4000 (Illumina, Inc.). Raw sequencing data was processed with the cellranger pipeline (version 2.1.0, 10X Genomics) and mapped to the mouse reference genome (mm10) to generate matrices of cancer cell transcripts by cell barcodes..