title: NextSeq 500 sequencing; RNA sequencing of tumor associated macrophages and T cells in clear cell renal cell carcinoma
design: RNA sequencing of tumor associated macrophages and T cells in clear cell renal cell carcinoma
library_construction_protocol: Primary ccRCC tissue samples were obtained from the University Health Network (UHN) Biospecimen Sciences Program and from the Cooperative Health Tissue Network (CHTN) from generously consenting patients under University Health Network Research Ethics Board approval.  Tumor samples were digested to generate single-cell suspensions as previously described (Gedye and Ailles, 2013). Cells were then viably frozen in 10% DMSO in D-SDCM complemented with 10% FBS. Viably frozen samples were resuscitated as previously described, incubated with the Zombie NIR dye (Biolegend) for dead cell exclusion for 15 min at room temperature, blocked with FcR blocking reagent (Miltenyi Biotech) for 10 min at 4 °C, and stained for 30 min at 4 °C with the antibodies indicated in Table S3. As shown in Figures S4C and S7B, cell subsets were sorted as CD14+/HLA-DR+/CD204-/CD38-/CD206- (Control) and CD14+/HLA-DR+/CD204+/CD38+/CD206- (M-5) , CD3+/CD8+/PD-1- (CD8+/PD-1- T cells) and CD3+/CD8+/PD-1+ (CD8+/ PD-1+ T cells) using a 100-μm nozzle on a FACS Aria III 5L Cell sorter. Cells were harvested in RPMI-FBS (10%) medium and immediately used for RNA purification using the PicoPure RNA Isolation Kit (ThermoFischer) according to manufacturer’s instructions 4 ng RNA was used to generate the sequencing libraries using the SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian (Takara Bio) and Nextera XT (Illumina) according to manufacturer’s instructions
attributes.tag: Experimental Factor: macrophage
attributes.value: cell type
attributes.tag: Experimental Factor: CD204-negative, CD38-negative
attributes.value: phenotype