Journal: Journal of the American Society of Nephrology : JASN

Article Title: Chromatin Conformation Links Distal Target Genes to CKD Loci

doi: 10.1681/ASN.2016080875

Figure Lengend Snippet: 4C-seq was used to study chromatin interactions, leading to the discovery of 304 CKD target genes in total. The 3D chromatin conformation of the DREs was studied in detail on the basis of the 4C template, which was generated by (A) fixing the chromatin structure, followed by (B) enzymatic (DpnI restriction enzyme) digestion of the fixed chromatin. (C) The digested chromatin was ligated into circular fragments in a diluted environment, after which (D) the chromatin was decrosslinked. (E) The circular DNA molecules followed another round of enzymatic (CvIQI restriction enzyme) digestion and ligation, after which the 4C-seq library was prepared with primers that target sequences in close proximity of the CKD susceptibility loci. (F) This library was sequenced to identify genes that were physically interacting with CKD susceptibility loci. (G) The 4C analysis was initially performed on 48 viewpoints on the basis of CKD-associated SNPs, of which, in total, 39 colocalized with active DREs on the basis of mapping with DNase hypersensitivity or H3K04me3 ChIP-seq datasets (33 in HRGECs and 34 in HRPTECs with partial overlap of SNPs). Of these 39 studied viewpoints, only 36 (31 in HRGECs and 34 in HRPTECs with partial overlap of viewpoints) were interacting with a total of 304 target genes with validated expression in the assessed cell types (overlap indicated in the Venn graph). These 304 genes were subsequently processed for genetic annotation to renal failure–associated traits in the OMIM database and the MGI database in addition to eQTL analysis in the GTEx portal database and pathway analysis using IPA software.

Article Snippet: The chromatin of the lysed cells was digested with the four-base cutter DpnI (NEB) followed by ligation in a heavily diluted environment with T4 ligase (Roche).

Techniques: Generated, Ligation, ChIP-sequencing, Expressing, Software

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Chromatin Conformation Links Distal Target Genes to CKD Loci

doi: 10.1681/ASN.2016080875

Figure Lengend Snippet: 4C-seq was used to study chromatin interactions, leading to the discovery of 304 CKD target genes in total. The 3D chromatin conformation of the DREs was studied in detail on the basis of the 4C template, which was generated by (A) fixing the chromatin structure, followed by (B) enzymatic (DpnI restriction enzyme) digestion of the fixed chromatin. (C) The digested chromatin was ligated into circular fragments in a diluted environment, after which (D) the chromatin was decrosslinked. (E) The circular DNA molecules followed another round of enzymatic (CvIQI restriction enzyme) digestion and ligation, after which the 4C-seq library was prepared with primers that target sequences in close proximity of the CKD susceptibility loci. (F) This library was sequenced to identify genes that were physically interacting with CKD susceptibility loci. (G) The 4C analysis was initially performed on 48 viewpoints on the basis of CKD-associated SNPs, of which, in total, 39 colocalized with active DREs on the basis of mapping with DNase hypersensitivity or H3K04me3 ChIP-seq datasets (33 in HRGECs and 34 in HRPTECs with partial overlap of SNPs). Of these 39 studied viewpoints, only 36 (31 in HRGECs and 34 in HRPTECs with partial overlap of viewpoints) were interacting with a total of 304 target genes with validated expression in the assessed cell types (overlap indicated in the Venn graph). These 304 genes were subsequently processed for genetic annotation to renal failure–associated traits in the OMIM database and the MGI database in addition to eQTL analysis in the GTEx portal database and pathway analysis using IPA software.

Article Snippet: The ligated samples were decrosslinked followed by a second digestion with the four-base cutter CvIQI (NEB).

Techniques: Generated, Ligation, ChIP-sequencing, Expressing, Software