title: Illumina HiSeq 4000 paired end sequencing; RNAseq profiling of tumor, tumor-draining-lymph nodes, blood and tumor-associated macrophages of MC38 mice treated with the PI3Kg inhibitor AZD3458 and a combination of AZD3458 and anti-PD-1
library_construction_protocol: Cell lines CT-26, 4T1 and MC-38 we purchased from ATCC. CT-26 (5x10^6 cells/mouse) or MC-38 (5x10^6 cells/mouse) tumor cells were implanted subcutaneously (s.c.) in the flank of female Balb/c and C57/Bl6 mice, respectively. 4T1 (5x10^6 cells/mouse) tumor cells were implanted orthotopically in mammary fat pad (o.t.) of female Balb/c mice or sub-cutaneous. At end of study tumor tissues were then transferred into the gentleMACS C Tube containing RPMI. Cells were liberated from tumors for downstream application using a mouse tumor dissociation kit from Miltenyi Biotec. and octodissociator (Miltenyi) according to manufacturerâ€™s instructions.  For RNA sequencing, total RNA was extracted using the RNeasy 96 Qiacube HT Kit (Qiagen), quality validated using nanodrop and Quantit RNA Assay Kit (Thermo Fisher). For Tumour and Lymph-Node libraries were made using TruSeq stranded mRNA kit and sequenced on the NovaSeq system (100bp, single end). For peripheral blood, libraries were made using TruSeq total RNA stranded with globin depletion. For sorted macrophages, libraries were made using clontech ultra low input mRNA kit.