Mus musculus
Protocols: MC38 were cultured in DMEM (Gibco) with 10% FBS (Gibco) and 4T1 were maintained in RMPI 1640 (Gibco) with 10% FBS at 37°C, 5% CO2. CT26 and A20 were cultured in RPMI-1640, 10% FCS, 1% L-Glutamine at 37°C , 7.5% CO2. A20 media was further supplemented with 0.05 mM 2-mercaptoethanol and 1% Pen Strep. Unless otherwise stated, mice were subcutaneously injected with 5x105 MC38 tumor cells in DPBS into the right flank while BALB/c mice were injected orthotopically with 1x105 4T1 tumor cells in the mammary fat pad using a 27 1⁄2g needle on Day 0 of study. For CT26 and A20, BALB/c mice were subcutaneously implanted into the right flank with 5x105 and 1x107 cells respectively. Tumors were measured using digital calipers measured 3x a week until the end of study or when tumors reached the ethical limit of 1500- 2000mm3 using calipers. RNA was extracted using Quick-RNA Microprep Kit (Zymo Research). Libraries for RNA sequencing were generated using the Takara SMART-Seq v4 PLUS Kit (Takara Bio; R400752) according to the manufacturer's protocol, with the following specificities. An input of 10ng of RNA per sample calculated from Agilent TapeStation DV200 values was used for cDNA generation. Post cDNA generation cleanup, cDNA for all samples were normalized to the cDNA concentration of the lowest yielding sample. Thus, cDNA input into library preparation was the same for all samples.