Qscript kit reagents

For RNA analysis of TAMs, WT, and IFNγR-deficient mice were injected subcutaneously in the hind flank with 1 × 10⁶ B16f10 melanoma cells. After 12 days, tumors were resected and processed into a single-cell suspension. Cells from tumors in three to four mice were pooled and sort isolated by flow cytometry based on the expression of CD45, CD11b, and F4/80. Sorted TAMs were then placed in Qiazol and RNA was isolated with a miRneasy kit from Qiagen. Primary cell and cell line cultures were lysed with Qiazol reagent prior to isolation of total RNA with a miRNeasy RNA isolation kit (Qiagen).
In total, 100–400 ng of RNA was utilized in a cDNA synthesis reaction with qScript kit reagents (Quanta Biosciences) and was diluted to a final volume of 100–200 µl. Quantitative PCR (qPCR) was performed in a Lightcycler LC480 (Roche) or a QuantStudio 6 (Thermo) utilizing Promega GoTaq qPCR master mix reagents or the equivalent, and qPCR primers listed in Supplementary Table 1. RT-qPCR data presented are representative of at least two independent experiments with at least three biological replicates per condition, and two to three technical replicate PCR reactions per sample. Gene symbols for target genes analyzed are listed in Supplementary Table 2.

Cells were lysed with Qiazol reagent prior to the isolation of total RNA with a miRNeasy RNA isolation kit (Qiagen, Germantown, MD, USA). An amount of 200–400 ng of RNA was utilized in a cDNA synthesis reaction with qScript kit reagents (Quanta Biosciences, Beverly, MA, USA) and was diluted to a final volume of 200 μL. Quantitative PCR (qPCR) was performed in a QuantStudio 6 (Thermo, Madison, WI, USA), utilizing Promega GoTaq qPCR master mix reagents, or the equivalent, and qPCR primers listed in Table S1. RT-qPCR data presented were representative of at least 2 independent experiments with at least 2 biological replicates per condition and 2 to 3 technical replicate PCR reactions per sample.