The downstream gene expression of Notch signal in tumor associated endothelial cells

To study the effects of Notch signal in tumor associated endothelial cells ,we conducted a RNA sequence assay. Briefly, TECs were isolatedas described above from LLC tumors 14 days after inoculation in 3 pairs of NICeCA and control mice. Total RNA was extracted using Trizol, and RNA integrity was evaluated using the Agilent 2200 Tape Station (Agilent Technologies, Santa Clara, California, USA) and each sample had the RINe above 7.0. rRNA was removed using the EpicentreRibo-Zero rRNARemoval Kit ( Illumina, San Diego, CA). Remaining RNA was fragmented into approximately 200bp fragments. Subsequently, the sample was subjected to first and second strand cDNA synthesis followed by adaptor ligation and enrichment with a low-cycle PCR usingTruSeq® RNA LT/HT Sample Prep Kit (Illumina). The purified library products were evaluated using the Agilent 2200 TapeStation and Qubit®2.0 (Life Technologies) and then diluted to 10 pM for cluster generation in situ on the HiSeq3000 pair-end flow cell followed by sequencing (2×150 bp) on HiSeq 3000. LLC tumor endothelial cells mRNA profile of endothelial specific Notch signal activation mice and control mice were generated by deep sequencing, in triplicate,using Illumina 3000