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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE111127 | GEO | 2019/02/26

REPOSITORIES: GEO

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Project description:Purpose: Pulmonary tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), has long been associated with the development of lung adenocarcinoma (LUAD) and sarcoidosis. These chronic lung diseases share histopathological similarities that challenge differential diagnosis. Methods: Human lung tissues were used for total RNA extraction with RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s protocol. RNA purity was assessed using a Nanodrop ND-2000 (Thermo Scientific), and each RNA sample had an A260:A280 ratio above 1.8 and an A260:A230 ratio above 2.0. RNA integrity was evaluated using the Agilent 2200 TapeStation (Agilent Technologies), and each sample had the RNA Integrity Number (RIN) above 7.0. The ribosomal RNAs (rRNAs) were removed using Ribo-Zero rRNA Removal Kits (Illumina). RNA libraries were then constructed by NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) and evaluated using the Agilent 2200 TapeStation and Qubit® 2.0 (Life Technologies). Sequencing was performed by RiboBio Co., Ltd. on an Illumina HiSeq 3000 machine with paired-end 150-bp reads. The adaptors and low quality bases assessed using FASTQC 0.11.3 were trimmed by Trimmomatic 0.39 using the following options: TRAILING:20, MINLEN:235 and CROP:235. Trimmed reads were then aligned to the ensembl 79 (GRCh38.p2) reference genome using STAR 2.4.2a. FeatureCounts 1.6.2 was subsequently employed to convert aligned short reads into read counts for each sample. Genes with less than ten counts in two or more samples were removed. The data were then analyzed using R 3.4.4 and DEseq2 1.18.1. Differentially expressed genes of each disease group were identified using Wald statistics test, with fold-change > 2 and Benjamini–Hochberg (BH)-adjusted P < 0.1 as compared to NC group. Results: These findings provide novel pathogenic links and molecular markers for better understanding and differential diagnosis of pulmonary TB, LUAD and sarcoidosis.

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

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