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Gene Expression in COP1-ETV1-ETV5-cJun deficient mouse brain

ABSTRACT: Total RNA was extracted from E18.5 brains with the olfactory bulbs removed using the MirVana miRNA isolation kit (Invitrogen). RNA concentration was determined in a NanoDrop 8000 (ThermoFisher) and RNA integrity using both 2100 Bioanalyzer and 2200 TapeStation (Agilent). Libraries were prepared from 1 μg of total RNA with TruSeq RNA Sample Preparation Kit v2 (Illumina). Library size was confirmed using 2200 TapeStation and High Sensitivity D1K screen tape (Agilent) and concentration was determined by Library quantification kit (KAPA). Libraries were multiplexed five per lane and then sequenced in a HiSeq2500 (Illumina) to generate 50 million paired end 75 bp reads. The "SAMPLE_ID" sample characteristic is a sample identifier internal to Genentech. The ID of this project in Genentech's ExpressionPlot database is PRJ0013017

ORGANISM(S): Mus musculus

PROVIDER: GSE111564 | GEO | 2018/11/14

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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Gene Expression in COP1-ETV1-ETV5-cJun deficient mouse brain

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