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A4eeb39d-5614-4251-aa1d-747339118ae2 - samples

ABSTRACT: Sequencing libraries were constructed according to standard procedures from 600 ng of tumor and paired constitutional DNA. WES was captured using Agilent SureSelect V5 (50 Mb), Clinical Research Exome (54 Mb) kit, SureSelect XT human All exon CRE version 1 or 2, or Twist Human Core Exome Enrichment System. Sequencing of subsequent libraries was performed using Illumina sequencers (Next-Seq 500 or Hiseq 2000/2500/4000) in 75 bp paired-end mode, aiming for a mean depth of coverage of 100x.

PROVIDER: EGAD00001010928 | EGA |

REPOSITORIES: EGA

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Project description:Non-coding regions of the genome contain cis-regulatory elements (CREs) that govern transcriptional activity critical for development and physiologic functions of major metabolic tissues, such as islets, adipose, liver, and skeletal muscle. Moreover, they contain the majority of genetic variants associated with risk of type 2 diabetes and quantitative alterations in related metabolic phenotypes. Here, we defined comprehensive, representative CRE maps of these major metabolic tissues in mice of both sexes using ATAC and pcHiC data and incorporating genetic variation of the 8 major Collaborative Cross founders and diet-response. Cross-species comparison of these representative maps with those from corresponding human tissues revealed conservation and functional preservation of ~28% of CREs in one or more major metabolic tissues. Importantly, this included an average of 700 (1.8%) cross-species concordant peaks that harbored genetic variants associated with T2D and related metabolic traits, including variants in the CAMK1D/CDC123, C2CD4A/B, and ADCY5 loci. Germline knock-out of the cross-species concordant CRE in ADCY5 yielded mice with impaired fasting glucose, demonstrating the importance of these functionally preserved CREs to glucose homeostasis and genetic risk of type 2 diabetes and progression. Together, the creation and cross-species comparison of comprehensive CRE maps in major metabolic tissues provides a critical resource for variant-to-function studies of type 2 diabetes and an array of related metabolic and cardiovascular traits and demonstrates the power of these mapping strategies to identify conserved metabolic cis-regulatory networks and enable data-driven creation of new preclinical models of diabetes and related metabolic diseases. Sample preparation: Promotor capture libraries were constructed via the following method. Crosslinked cells were lysed, digested with DpnII, ends filled in using biotin-14-dATP followed by overnight proximity ligation. Ligated samples underwent crosslink reversal, DNA extraction with proteinase K, and biotin removal from unligated fragments. DNA was fragmented to 500-600 bp, column purified, and biotinylated fragments pulled down. Biotinylated fragments were converted into Illumina compatible libraries using the SureSelect XT HS2 DNA System and a custom SureSelect probe set (design ID S3346176) (Agilent Technologies) according to the manufacture’s protocol with the following exception: pre-capture PCR was performed as four 50 ul reactions that were recombined during clean-up. The quality and concentration of the libraries were assessed using the High Sensitivity D5000 ScreenTape (Agilent Technologies) and Qubit dsDNA HS Assay (ThermoFisher), respectively, according to the manufacturers’ instructions. Libraries were sequenced 150 bp paired end on an Illumina NovaSeq 6000.

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A4eeb39d-5614-4251-aa1d-747339118ae2 - samples

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