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RNA-seq analysis of the spinal cord cells in scramble microPEP(Scr) or microPEP155(P155) treated EAE mouse model

ABSTRACT: The goals of this study are to analyze the DEGs of the spinal cord cells in scramble microPEP(Scr) or microPEP155(P155) treated EAE mouse model. Methods: mononuclear cells of the spinal cord were isolated using Percol™ gradient centrifugation, washed twice with PBS, and total RNA was extracted with RNAiso Plus (Takara Bio) and purified with magnetic oligo (dT) beads after denaturation. Purified mRNA samples were reverse transcribed into fragmented DNA samples and adenylated at the 3’ ends. Adaptors were ligated to construct a library. DNA was quantified by Qubit (Invitrogen). After cBot cluster generation, DNA samples were then sequenced by an Illumina HiSeq X Ten SBS instrument from Genergy Bio (Shanghai). Raw data were converted into Fastq format and transcript per million fragments mapped (FPKM) was calculated and log2 transformed with Cuffnorm. Differential gene transcripts were analyzed with DESeq and enriched for the GO/Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Results:Gene Ontology (GO) pathways analysis of RNA-seq data revealed that genes downregulated by P155 treatment were mainly involved in the immune respones signaling pathway.

ORGANISM(S): Mus musculus

PROVIDER: GSE142710 | GEO | 2019/12/31

REPOSITORIES: GEO

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Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to find the potential molecular mechanism of traditional Chinese medicine in the treatment of spinal cord injury Methods: Total RNA was isolated and purified using TRIzol reagent following the manufacturer's procedure. The RNA amount and purity of each sample was quantified using NanoDrop ND-1000 . The RNA integrity was assessed by Bioanalyzer 2100 with RIN number >7.0, and confirmed by electrophoresis with denaturing agarose gel. Poly (A) RNA is purified from 1μg total RNA using Dynabeads Oligo (dT)25-61005 using two rounds of purification. Then the poly(A) RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module under 94℃ 5-7min. Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase , which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I, RNase H and dUTP Solution . An A-base is then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contains a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single- or dual-index adapters are ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products are amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. The average insert size for the final cDNA library was 300±50 bp. At last, we performed the 2×150bp paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 following the vendor's recommended protocol. Results:In this study, we obtained: chromosome: 23, Genes (G): 32623, Transcripts (T): 40808, GO Annoated: 20795, KEGG Annoated: 8299; in further pathway analysis, it was shown that PI3K-AKT signaling pathway may be the underlying molecular mechanism. Conclusions: Our study represents the first detailed analysis of the spinal cord transcriptome generated by RNA-seq technology with biological replicates. Our results suggest that Modified Erxian decoction may mediate spinal cord repair through the PI3K-AKT pathway.

Project description:We have investigated the process of disease-induced functional perturbation and the related transcriptional changes occurring in thoraco-lumbar spinal cord extracted from Sprague-Dawley rats heterozygous for the G93A SOD1 gene mutation (Emerging Model 2148 Het Male, Taconic USA; Wyeth and Amyotrophic Lateral Sclerosis Association 2002) using spinal cord from wild type females littermates as reference tissues. Rats were obtained from a breeding project at Taconic Breeding Services (USA). We have applied large-scale gene expression analysis to define the pattern or transcriptional changes occurring in spinal cord from the G93A SOD1 rat model from a pre-symptomatic stage, at disease onset and at end-stage disease, using Bead Array analysis (Illumina, San Diego, USA). We have pooled spinal cord from N:5 transgenic rats for each of the time points considered, using the same pools of spinal cord from sex and age-matched WT rats as reference. In this specific project, the aim was to obtain a gene ontology (GO) pathway analysis of the transcriptional changes induced by the G93A SOD1 mutation in rat spinal cord. Hence, we have opted for a sample pooling strategy, well aware that in so doing, we would not obtaineed information about individuals genes variation across the samples in study but an overall view of the activation of multi-genes molecular signals. Total RNA was isolated from the spinal cords of mutant (G93A SOD1 gene mutation) female rats sacrificed at a pre-symptomatic stage (10-week old), at disease onset and at end stage disease and from age and sex-matched wild type (WT) littermates. RNA samples obtained from spinal cord extracted from rats of the same genetic types and sacrificed at the same time points (e.g. 5 RNA samples from mutant spinal cord from end-stage rats; 5 RNA samples from mutant spinal cord from rats at disease onset; 5 RNA samples from mutant pre-symptomatic rats and 5 RNA samples from spinal cord obtained from age-matched WT rats sacrificed at each of the 3 time points) were pooled and used for gene expression analysis and Ontology analysis of the expression profiles.

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RNA-seq analysis of the spinal cord cells in scramble microPEP(Scr) or microPEP155(P155) treated EAE mouse model

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