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Next Generation Sequencing to study Thalidomide mechanism in Cutaneous Lupus

ABSTRACT: Next Generation Sequencing to study Thalidomide mechanism in Cutaneous Lupus

PROVIDER: PRJNA681843 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR13175159_1.fastq.gz	Fastqsanger.gz
	SRR13175159_2.fastq.gz	Fastqsanger.gz
	SRR13175160_1.fastq.gz	Fastqsanger.gz
	SRR13175160_2.fastq.gz	Fastqsanger.gz
	SRR13175161_1.fastq.gz	Fastqsanger.gz

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Next Generation Sequencing to study Thalidomide mechanism in Cutaneous Lupus

Project description:Thalidomide has been shown to be effective in patients with refractory cutaneous lupus erythematosus (CLE). However, its use is still limited by its potential severe side-effects. We conducted an RNA-sequencing study using CLE skin biopsies before and after thalidomide treatment to discover its mechanism of action. Methods: Total mRNA from skin biopsies from 10 CLE patient have been processed by RNA-seq in duplicate using Illumina Hiseq 2000 sequencing platform version 3. Application was stranded mRNA-Seq, paired-end and read length was 75bp. The sequence reads that passed quality filters were analyzed at the transcript isoform level using STAR program, RSEM program and DESeq2. Mapping was done with STAR (version 2.5.2a), quantification by RSEM (1.2.28) and differential analysis by DESeq. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to human genome (hs37d5). Average un-mapped was between 11,40-7,05 %, average unique was 80.48-76,62% and average alignment insert size was 170,00-155,00 in the 20 samples. Conclusions: Plausibles mechanisms of action for thalidomide in lupus cutaneous were discovered using this study; however, in vitro and in vivo experiments would be performed to demostrate them.

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Project description:Lesional skin biopsies were taken from patients with active, untreated lupus skin disease (chronic discoid lupus erythematosus, CDLE, n=6; subacute cutaneous lupus erythematosus, SCLE, n=5). Healthy control specimens (HC) were obtained from healthy skin of 5 patients undergoing plastic surgery. In every case, two 4mm punch biopsies were taken. One was flash-frozen in liquid nitrogen and afterwards processed for mRNA isolation. The second biopsy was fixed in 5% formalin solution overnight, and was proceeded for histological investigation.The one-color Agilent 60-mer oligo microarray (Agilent, Santa Clara, CA) was used for gene expression analyses. Statistical analyses were performed using the Agilent Feature Extraction Softwareâ¢ and the Rosetta Resolverâ¢ gene expression data analysis system. The presented gene list (Table S1) includes normalized sample/ control log10-ratios (expression > 2-fold enhanced, p<0.01).

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