RNA Sequencing in CD11b cells for Quantitative Analysis of WT and myeloid specific NRP2 Knock out transcriptomes in subcutaneous pancreatic tumors

The purpose of the study is to identify the alterations in transcriptome (RNA Seq) in CD11b+ myeloid cells isolated from WT and monocytes/macrophage specific Neuropilin2 (NRP2) Knock out mice harboring subcutaneous pancreatic cancer tumors. The main goal is to identify the intrinsic changes in gene signature occuring in myeloid cells in NRP2 deleted conditions in tumor that indicates the role of NRP2 in regulating phagocytosis/efferocytosis and immune responses in myeloid cells in tumors. Mouse pancreatic cancer cell lines were subcutaneously implanted into NRP2f/f i-Cre mice. Once tumors became palpable, the test group animals were injected with Tamoxifen to specifically knock out NRP2 from monocytes/macrophages. Tumors were harvested at 15days and CD11b cells were isolated using magnetic beads. RNA was isolated using trizol and RNA Seq performed for analysis of differentially expressed transcriptomes in WT and in myeloid specific NRP2 KO conditions. We had n=3 animals in each control and test groups. Since the RNA isolated from each individual animal was less, we pooled RNA from n=3 animals. We validated expression of some of the differentially altered genes in seperate biological replicates by RT-PCR analysis.