RNAseq for B16-OVA and 4T1 murine cancer cell lines
After harvesting cells, we washed 3x with PBS (phosphate buffer saline, Gibco DPBS, no calcium, no magnesium, 14190-144, Fisher Scientific P/N 12559069), centrifuged at 2000 g, discarded supernatant and snap froze the cell pellets until further use. After thawing the cell pellets, aliquots of about 5 million cells were taken separately for DNA/RNA extraction and proteomics analysis. B16/OVA cells were grown (24-36h doubling period) in RPMI 1640 (cat. No. 42401 GIBCO) medium supplemented with 10% heat inactivated fetal bovine serum (cat. No. 10500 GIBCO), 1% Penicilin-Streptomycin (cat. No. 15140 GIBCO) and 1% glutamine (GlutaMAX cat. No. 35050 GIBCO). B16/OVA cells were kept in selection with 10% Geneticin Selective Antibiotic (G418 Sulfate) (cat. No. 10131 GIBCO) and medium was changed every two/three days.  Murine mammary carcinoma 4T1 cells were grown (12h doubling period) in RPMI 1640 high glucose (cat. No. A1049101 GIBCO) medium supplemented with 10% heat inactivated fetal bovine serum (cat. No. 10500 GIBCO) and 1% Penicilin-Streptomycin (cat. No. 15140 GIBCO).  All cell lines were cultured at 37°C in 5% CO2 in a humidified atmosphere. All cells were tested for Mycoplasma contamination using MycoAlert kit (Lonza, cat. LT07-118).  Qiagen all prep DNA/RNA extraction kit (Qiagen, cat. No. 80284) was used to extract DNA and RNA from the mouse cancer samples according to the manufacturer's instructions. Illumina TruSeq strand specific RNA libraries with RiboZero Gold treatment was used to prepare the tumor RNA samples. RIN values were verified by Bioanalyzer 2100 (Agilent) and they were above 7 for all RNA samples.