GSM4023824: FF99WT_B; Mus musculus; RNA-Seq
Dissected primary tumors were cut and minced and then incubated in DMEM/F12 supplemented with 5% FBS, 1x antibiotic-antimycotics, gentamycin (20 µg/mL) (all from Invitrogen), collagenase (300 units/mL), and hyaluronidase (100 units/mL) (Worthington Biomedical) for 2 hours at 37 oC. They were then centrifuged at 400 g for 5 minutes, and the pellets were washed with PBS, and suspended in 0.05% trypsin/0.025% EDTA (Invitrogen) before incubated for 5 minutes at 37 oC. An equal volume of DMEM/F12 containing 5% FBS and DNase I (20 units/ml, Roche) was then added to stop the digestion.  After filtering through a 40µm nylon mesh (BD Biosciences), the cells were collected by centrifugation at 400g for 5 min and re-suspended in ACK lysis buffer (Invitrogen) to lyse red blood cells. Tumor single-cell suspensions were incubated with CD24-PE (553262, BD Biosciences), CD31-APC (102410, Biolegend), CD45-APC (103112, Biolegend), Ter119-APC (116212, Biolegend) according to manufacturer's instructions for 20 minutes at 40C. Following that, cells were then rinsed and resuspended in HBSS before sorting or analysis by FACSAria or FACSCanto instruments (BD Biosciences). For isolation of CD14+ cells, CD14-APC/Cy7 (123317, Biolegend) antibody was used. Single-cell cDNA libraries were prepared using the 10x Chromium single-cell kit (3' gene expression platform version 2 chemistry) according to manufacturer's instructions for each tumor separately.