RNA sequencing analysis of NK cells, T cells and tumor from matched soft tissue sarcoma and peripheral blood

Using fluorescence-activated cell sorting, we isolated blood and tumor-infiltrating CD3-CD56+ NK and CD3+ T cells and CD45- viable tumor cells from STS patients undergoing surgery. We then evaluated differential gene expression (DGE) of these purified populations with RNA sequencing analysis The collection of matched whole blood and tumor specimens was approved by the IRB at the University of California, Davis. Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using a density gradient centrifugation, followed by red blood cell lysis. Tumor tissue from patients undergoing STS resection was collected in sterile RPMI-1640, diluted with PBS and passed through a 70 µm filter, and then subjected to red blood cell lysis. Human cells were washed with PBS and staining buffer, and then incubated with Fc receptor blocking solution for 15 minutes. Cells were stained with the following fluorochrome-conjugated monoclonal antibodies: CD3-FITC, CD56-PE, CD45-BV510 and CD8-BV785. Live/dead staining was performed using Fixable Viability Dye 780. Data were acquired using a BD LSRFortessa flow cytometer and analyzed using FlowJo Software. Fluorescence-associated cell sorting (FACS) was performed using a BD inFlux cell sorter. Cells were sorted for blood and intra-tumoral NK cells (live CD45+CD3-CD56+), blood and intra-tumoral T cells (live CD45+CD3+CD56-), and tumor cells (live CD45-). Following FACS isolation, cells were cryopreserved and stored in liquid nitrogen for subsequent RNA extraction and sequencing. Cells were processed for RNA isolation using the Total RNA Purification Plus Micro Kit. RNAseq libraries were prepared by the University of California, Davis, Bioinformatics Core. Gene expression profiling was carried out using a 3'-Tag-RNA-Seq protocol. Up to forty-eight libraries were sequenced per lane on an Illumina HiSeq 4000 sequencer with single-end 100 base pair reads. Differential gene expression analysis was performed with the DESeq2 package for R. The Benjamini-Hochberg procedure was used to adjust for False Discovery Rate (FDR).