Agilent bioanalyzer tapestation analysis

Total RNA was isolated using RNeasy Mini Kit (Qiagen) from mouse BMDMs treated with either 4T1 CM, CM with 0.5 μM ruxolitinib or CM with 0.5 μM ruxolitinib and 0.8 μM IKK-16. Samples were submitted in triplicate to the University of Minnesota Genomics Center. Library preparation was done through TruSeq Stranded mRNA Library Preparation and quality control was performed using Agilent Bioanalyzer/TapeStation analysis. Samples were multiplexed in one lane of NovaSeq S2 and 150 bp paired-ends were sequenced (20 M reads/sample). All samples passed quality control, containing > 500 ng of RNA and having an RIN above 8. Individual gene expression plots were obtained by calculating counts per million (CPM) for each gene. For downstream analysis, genes with LogFC > 0.5 and FDR < 0.05 were selected.

RNA was extracted from unstained formalin-fixed, paraffin-embedded (FFPE) slides. We obtained five 5 mm-thick unstained FFPE slides of each patient (9 CR and 3 LR). The tissues from five FFPE sections were combined and used for the simultaneous isolation of total RNA and DNA with Quick-DNA/RNA FFPE kit (R1009) from Zymo Research, Costa Mesa, CA according to the manufacturer’s protocol. RNA was treated with DNase1 in the columns. The concentration and purity of RNA were assessed using Nanodrop spectrophotometer (Thermo Scientific, Waltham, MA). Additional QA/QC of the samples was conducted in the Genomics Core Laboratory at the University of Minnesota using Agilent BioAnalyzer/TapeStation Analysis. Assays were performed with 20 ul volume, with a minimal of total 400 ng of RNA.

Total RNA was isolated using RNeasy Mini Kit (Qiagen) from mouse BMDMs treated with either 4T1 CM, CM with 0.5 μM ruxolitinib or CM with 0.5 μM ruxolitinib and 0.8 μM IKK-16. Samples were submitted in triplicate to the University of Minnesota Genomics Center. Library preparation was done through TruSeq Stranded mRNA Library Preparation and quality control was performed using Agilent Bioanalyzer/TapeStation analysis. Samples were multiplexed in one lane of NovaSeq S2 and 150 bp paired-ends were sequenced (20 M reads/sample). All samples passed quality control, containing > 500 ng of RNA and having an RIN above 8. Individual gene expression plots were obtained by calculating counts per million (CPM) for each gene. For downstream analysis, genes with LogFC > 0.5 and FDR < 0.05 were selected.