Unknown,Transcriptomics,Genomics,Proteomics

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RNA sequencing of a C57BL/6J-DBA/2J (B6.D2) congenic mouse and C57BL/6J (B6) wild-type littermates reveals cis and trans differences in gene expression

ABSTRACT: We previously utilized interval-specific congenic lines derived from C57BL/6J (B6) and DBA/2J (D2) alleles to fine map a quantitative trait locus (QTL) influencing methamphetamine (MA)- induced locomotor activity. We identified a 0.23 MB critical interval on chromosome 11 containing only two protein coding genes, Rufy1 and Hnrnph1. Notably, Rufy1 contains three missense SNPs and Hnrnph1 contains 1 SNP near the 5â UTR. In an effort to identify the molecular mechanisms that bridge genetic variation with behavior, we conducted transcriptome analysis via mRNA sequencing (RNA-seq) in a B6.D2 congenic line (chr.11: 50-60 Mb) that captures the QTL. There was an overrepresentation of cis-regulated, differentially expressed genes within the congenic interval (4 out of 92 differentially expressed genes; FDR < 0.05) and widespread genomic regulation on all autosomes. For this study, 3mm punches from both striatal hemispheres were collected and pooled from 16 mice (8 controls and 8 B6.D2 congenics), and RNA was extracted and prepared for cDNA library preparation using the Illumina TruSeq (oligo-dT; 50bp single-end reads). Samples 1-16, consisting of alternating B6 and B6.D2 congenics were run in four lanes total, with samples 1-8 run in duplicate across lanes 5 and 6 and samples 9-16 acorss lanes 7 and 8, respectively. Raw samples provided are labeled with the "CB-" prefix, followed by the sample number and the lane that it was run in is described at the tail end as "_L00#" with # being either lane 5, 6, 7 or 8. In this study, all odd samples (e.g. CB-1,3,5 etc.) are C57BL/6J littermate controls, while all even samples are B6.D2 congenics. Note that the lane 7 replicate for Sample CB-13 (CB-13_L007.fastq.gz) was not processed due to technical difficulties with the file.

ORGANISM(S): Mus musculus

SUBMITTER: Camron Bryant

PROVIDER: E-GEOD-66366 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: The goal of the present study is to provide an independent assessment of the retinal transcriptome signatures of the C57BL/6J (B6) and DBA/2J (D2) mice and to enhance existing microarray datasets for accurately defining the allelic differences in the BXD recombinant inbred strains. Methods: Retinas from both B6 and D2 mice (3 of each) were used for the RNA-seq analysis. Transcriptome features were examined for both strains. Differentially expressed genes between the 2 strains were identified and bioinformatic analysis was performed to analyze the transcriptome differences between B6 and D2 strains, including Gene ontology (GO) analysis, Phenotype and Reactome enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The RNA-seq data were then directly compared with one of the microarray datasets (DoD Retina Normal Affy MoGene 2.0 ST RMA Gene Level Microarray Database) hosted on GeneNetwork (www.genenetwork.org). Results: RNA-seq provided an in-depth analysis of the transcriptome of the B6 and D2 retina with a total of more than 30,000,000 reads per sample. Over 70% of the reads were uniquely mapped, resulting in a total of 18,100 gene counts for all 6 samples. 1,665 genes were differentially expressed, with 858 of these more highly expressed in B6 and 807 more highly expressed in D2. Several molecular pathways were differentially active between the two strains, including the retinoic acid metabolic process, endoplasmic reticulum lumen, extracellular matrix organization, and PI3K-Akt signaling pathway. The most enriched KEGG pathways were the pentose and glucuronate interconversions pathway, the cytochrome P450 pathway, protein digestion and absorption pathway and the ECM-receptor interaction pathway. Each of these pathways had a more than 4-fold enrichment. The DoD normal retina microarray database provided expression profiling for 26,191 annotated transcripts for B6 mouse, D2 mouse and 53 BXD strains. A total of 13,793 genes in this microarray dataset were comparable to the RNA-seq dataset. For both B6 and D2, the RNA-seq data and microarray data were highly correlated with each other (Pearson's r = 0.780 for B6 and 0.784 for D2). Our results suggest that the microarray dataset can reliably detect differentially expressed genes between the B6 and D2 retinas, with a positive predictive value of 45.6%, and a negative predictive value of 93.6%. Examples of true positive and false positive genes are provided. Conclusions: Retinal transcriptome features of B6 and D2 mouse strains provide a useful reference for a better understanding of the mouse retina. Generally, the microarray database presented on GeneNetwork shows good agreement with the RNA-seq data, while we note that any allelic difference between B6 and D2 should be verified with the latter.

Project description:Background and Aims: In the interleukin-10-deficient (Il10-/-) mouse model of IBD, 10 quantitative trait loci (QTL) have been shown to be associated with colitis susceptibility by linkage analyses on experimental crosses of highly susceptible C3H/HeJBir (C3Bir)-Il10-/- and partially resistant C57BL/6J (B6)-Il10-/- mice. The strongest locus (C3Bir-derived cytokine deficiency-induced colitis susceptibility [Cdcs]1 on Chromosome [Chr] 3) controlled multiple colitogenic subphenotypes and contributed the vast majority to the phenotypic variance in cecum and colon. This was demonstrated by interval-specific Chr 3 congenic mice wherein defined regions of Cdcs1 from C3Bir or B6 were bred into the IL-10-deficient reciprocal background and altered the susceptible or resistant phenotype. Furthermore, this locus likely acts by inducing innate hypo- and adaptive hyperresponsiveness, associated with impaired NFΚB responses of macrophages. The aim of the present study was to dissect the complexity of Cdcs1 by further development and characterization of reciprocal Cdcs1 congenic strains and to identify potential candidate genes in the congenic interval. Material and Methods: In total, 15 reciprocal congenic strains were generated from Il10-/- mice of either C3H/HeJBir or C57BL/6J backgrounds by 10 cycles of backcrossing. Colitis activity was monitored by histological grading. Candidate genes were identified by fine mapping of congenic intervals, sequencing, microarray analysis and a high-throughput real-time RT-PCR approach using bone marrow-derived macrophages. Results: Within the originally identified Cdcs1-interval, three independent regions were detected that likely contain susceptibility-determining genetic factors (Cdcs1.1, Cdcs1.2, and Cdcs1.3). Combining results of candidate gene approaches revealed Fcgr1, Cnn3, Larp7, and Alpk1 as highly attractive candidate genes with polymorphisms in coding or regulatory regions and expression differences between susceptible and resistant mouse strains. Conclusions: Subcongenic analysis of the major susceptibility locus Cdcs1 on mouse chromosome 3 revealed a complex genetic structure. Candidate gene approaches revealed attractive genes within the identified regions with homologs that are located in human susceptibility regions for IBD. Experiment Overall Design: Bone marrow derived macrophages (BMDM) of colitis susceptible C3Bir-Il10-/- and colitis resistant B6-Il10-/- as well as the Il10-/- reciprocal congenic strains CB-R1 (C3Bir genetic background carrying a long congenic chromosome 3 element containing Cdcs1 from B6, rendering this formerly susceptible background resistant) and BC-R3 (B6 genetic background that carries the Cdcs1-region of C3Bir) were cultured and stimulated with flagellin or left unstimulated. BMDM were obtained from 3 male mice per genotype and cultured in polystyrene 6-well culture plates. Before RNA-isolation, 3 wells per plate were stimulated with Cbir1 flagellin (and subsequently pooled for RNA isolation), the other 3 wells left unstimulated (and also pooled). In total, these experiments were replicated three times in order to perform microarray analyses in triplicates.

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RNA sequencing of a C57BL/6J-DBA/2J (B6.D2) congenic mouse and C57BL/6J (B6) wild-type littermates reveals cis and trans differences in gene expression

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