Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNASeq from colonic tissue of naïve IEC-Bmal1-/- mice and Bmal1flox controls over the course of a day

ABSTRACT: Circadian clocks are important for gut health. This experiment aimed to determine the role of core clock gene Bmal1. Mice were generated with Bmal1 selectively deleted in Villin-expressing cells (predominantly IECs). Colon tissue was harvested from Villin-Bmal1-/- and Bmal1flox controls over the course of the day at zeitgeber time (ZT)0, ZT6, ZT12 and ZT18. Colon tissue was homogenised in lysing MatrixD tubes (MP biomedical) containing TRIzol (Invitrogen), using a bead mill homogeniser (Fisherbrand). RNA was extracted with chloroform and aqueous phase was precipitated with isopropyl alcohol. Supernatant was washed with 70% ethanol and the RNA pellet was resuspended in nuclease-free water. Sequencing library preparation with TruSeq Stranded mRNA assay (Illumina) and sequencing were performed by the University of Manchester Genomic Technologies Core Facility. Multiplexed libraries were analysed by paired-end sequencing on NovaSeq 6000 (Illumina). Quality control was performed with Fastqc (v0.11.3) and FastqScreen (v0.14.0). Reads were trimmed using BBDuk from BBMap (v38.96) and reads were mapped to the mouse genome (mm39/vM30) using STAR (v2.7.10a).

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Mus musculus

SUBMITTER: Tom Butler

PROVIDER: E-MTAB-14341 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Goal: Since disease susceptibility of the intestine exhibits an anatomical bias, we propose that the chromatin landscape, especially the site-specific epigenetic differences in histone modification patterns throughout the longitudinal axis, contributes to a differential response to chemoprotective agents. Method: We assessed the chromatin structure associated with gene expression profiles in the rat proximal and distal colon by globally correlating chromatin immunoprecipitation next-generation sequencing analysis (ChIP-Seq) with mRNA transcription (RNA-Seq) data. Crypts were isolated from the proximal and distal colonic regions from rats maintained on a semi-purified diet, and mRNA gene expression profiles were generated using RNA-Seq. The remaining isolated crypts were paraformaldehyde-crosslinked and chromatin immunoprecipitated using antibodies against H3K4me3, H3K9me3, and RNA Polymerase II. Results/Conclusion: Globally, RNA-Seq results indicated that 9,866 genes were actively expressed, of which 540 genes were differentially expressed between the proximal and distal colon. With regard to differentially expressed genes, a high correlation was observed between H3K4me3-Seq and RNA-Seq data, with 96% of the canonical pathways being similarly affected in the H3K4me3-Seq and RNA-Seq data sets. Gene ontology analysis indicated that colonic crypt location significantly impacted both chromatin and transcriptional regulation of genes involved in cell transformation, lipid metabolism, lymphatic development and immune cell trafficking. Gene function analysis indicated that the PI3-Kinase signaling pathway was regulated in a site-specific manner, e.g., pathway proto-oncogenes, c-Jun, c-Fos and ATF, were up-regulated in the distal colon. Middle and long non-coding RNAs (lncRNAs) were also detected in the colon, including select lncRNAs formerly only detected in the rat nervous system. In summary, distinct combinatorial patterns of histone modifications exist in the proximal versus distal colon. These site-specific differences may explain the differential effects of chemoprotective agents on cell transformation in the ascending (proximal) and descending (distal) colon. Examination of mRNA profiles and 2 different histone modifications (H3K4me3 and H3K9me3) in 2 tissue types (proximal and distal colon).

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RNASeq from colonic tissue of naïve IEC-Bmal1-/- mice and Bmal1flox controls over the course of a day

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