GSM9597674: SP-neg; Mus musculus; RNA-Seq
Mice bearing ZsGreen-expressing 6694c2 tumors were treated as described above. 48 hours after treatment, tumors, spleens, and tumor-draining lymph nodes were harvested from mice. Spleens and lymph nodes were macerated. Tumors were macerated and digested in collagenase IV (Sigma #C5138) and soybean trypsin inhibitor (Life Technologies #17075029). Tumor chunks were removed via filtration with a 40 μM filter, and the resulting single cell suspension was washed with PBS. Part of the tumor, lymph node, and spleen sample was taken for flow cytometry analysis. The rest of the samples were hashed, sorted based on ZsGreen expression on a BD FACS Aria, and analyzed via single-cell sequencing analysis. 6694c2 and B16F10 tumor cells were treated with IFNγ (100 ng/mL) (Peprotech #315-05), IFNβ (1000 U/mL) (BioLegend #581302), and STING agonist (225 ug/mL) in culture for 24 hrs. Cells were harvested and RNA isolated with a QIAGEN RNAeasy Plus Mini Kit (#74134). RNA was sent to Genewiz for paired-end RNA sequencing. Cells were then loaded onto a Chromium X with the Single Cell K Chip (PN-2000182), the Chromium Next GEM Single Cell 5ʹ GEM Kit v2 reagents, and beads (PN-1000244). Library preparation was performed with the Library Construction Kit (PN-1000190). Libraries were pooled and sequenced on an Illumina NovaSeq 6000 instrument with 2x150bp sequencing (Azenta Life Sciences).