Project description:ChIP-seq has become the method of choice for studying functional DNA-protein interactions on a genome wide scale. The method is based on co-immunoprecipitation of DNA binding proteins with formaldehyde cross-linked DNA, followed by deep sequencing of immunoprecipitated chromatin fragments, allowing for the identification of binding sites with high accuracy [1-5]. Traditionally, genome-wide tiling path microarrays were used for the detection of specifically immunoprecipitated DNA fragments, but current next-generation sequencers now generate sufficient data to assay multiple samples in a single sequencing run, making this method more time and cost effective compared to microarray-based approaches [2]. However, due to limitations in read length of next generation sequencing technologies (50-76bp), it is not possible to sequence the complete length of immunoprecipitated DNA fragments which can be up to 2kb long reflecting biology in case of big protein complexes. As a consequence only the ends of the immunoprecipitated DNA fragments are sequenced. Deconvolution of sequencing reads mapping to the positive and negative strand is required to identify the real DNA binding site [4-9]. This limitation is most obvious in case of large complex regions where multiple binding sites of various regulatory elements are clustered close to each other. Deconvolution of such regions and the exact identification of individual binding positions is very challenging [4]. Similar problems can occur when studying histone positioning in cases where the length of ChIP fragments is bigger than the average distance of 2 neighboring nucleosomes. In addition frequently only narrow size range of immunoprecipitated fragments is selected for sequencing and thus possible bias towards binding regions within the selected range can be expected by loosing larger fragments possible originated from protein complexes interacting with DNA. To circumvent these complications, we modified the procedure for the preparation of ChIP-seq samples by introducing an additional extensive fragmentation round after isolation of immunoprecipitated chromatin to generate 70-110 bp long DNA fragments. For testing, we choose Tcf4 protein which is a well-studied downstream element of the Wnt-pathway for which the genome wide binding site profile is known was already known from ChIP-on-CHIP experiments [10]. Using the modified approach, we were able to identify Tcf4 binding sites at near nucleotide resolution without computational deconvolution. Furthermore, high-resolution information on genome-wide binding site regions allowed for the identification of potential novel Tcf4 co-factors as well as target genes. 5 samples + 3 input samples

Project description:ChIP-seq has become the method of choice for studying functional DNA-protein interactions on a genome wide scale. The method is based on co-immunoprecipitation of DNA binding proteins with formaldehyde cross-linked DNA, followed by deep sequencing of immunoprecipitated chromatin fragments, allowing for the identification of binding sites with high accuracy [1-5]. Traditionally, genome-wide tiling path microarrays were used for the detection of specifically immunoprecipitated DNA fragments, but current next-generation sequencers now generate sufficient data to assay multiple samples in a single sequencing run, making this method more time and cost effective compared to microarray-based approaches [2]. However, due to limitations in read length of next generation sequencing technologies (50-76bp), it is not possible to sequence the complete length of immunoprecipitated DNA fragments which can be up to 2kb long reflecting biology in case of big protein complexes. As a consequence only the ends of the immunoprecipitated DNA fragments are sequenced. Deconvolution of sequencing reads mapping to the positive and negative strand is required to identify the real DNA binding site [4-9]. This limitation is most obvious in case of large complex regions where multiple binding sites of various regulatory elements are clustered close to each other. Deconvolution of such regions and the exact identification of individual binding positions is very challenging [4]. Similar problems can occur when studying histone positioning in cases where the length of ChIP fragments is bigger than the average distance of 2 neighboring nucleosomes. In addition frequently only narrow size range of immunoprecipitated fragments is selected for sequencing and thus possible bias towards binding regions within the selected range can be expected by loosing larger fragments possible originated from protein complexes interacting with DNA. To circumvent these complications, we modified the procedure for the preparation of ChIP-seq samples by introducing an additional extensive fragmentation round after isolation of immunoprecipitated chromatin to generate 70-110 bp long DNA fragments. For testing, we choose Tcf4 protein which is a well-studied downstream element of the Wnt-pathway for which the genome wide binding site profile is known was already known from ChIP-on-CHIP experiments [10]. Using the modified approach, we were able to identify Tcf4 binding sites at near nucleotide resolution without computational deconvolution. Furthermore, high-resolution information on genome-wide binding site regions allowed for the identification of potential novel Tcf4 co-factors as well as target genes.

Project description:Ischemia-reperfusion injury during liver transplantation is responsible for early allograft dysfunction (EAD) and failure, both of which are associated with a high risk of morbidity and mortality in the recipient. The purpose of this study was to study major transcriptional alterations in livers procured from different types of human liver donors in order to identify genetic profiles predictive of post-implantation function. We have analyzed samples form living donors (LD), donors after cardiac death (DCD), donors after brain death, with subsequent post-implantation EAD in the recipient (DBD-EAD); and donors after brain death without EAD (DBD). Two samples were obtained from each donor: sample A was taken immediately before cold perfusion (baseline) and sample B 2h after portal reperfusion. We identified clear differences in gene expression patterns according to donor source. Both samples A and B from DBD-EAD and DCD demonstrated over-expression of pro-apoptotic and inflammatory transcripts. However, in DBD and LD, expression of these genes was low at baseline and rose only after reperfusion. DBD and LD demonstrated the greatest increase in overall genetic expression after reperfusion when sample B was contrasted with A, indicating less baseline graft injury in these two groups. Grafts from LD were characterized by activation of transcripts related to anti-ischemic and regenerative processes and fewer pro-inflammatory gene transcripts. This transcriptional events occurring in liver allografts could allow for the prediction of post-transplant function. Pro-inflammatory and ischemic transcriptional changes in the grafts are directly related to donor type and may be useful targets for the development of future therapeutic strategies. The complete database comprised the expression for samples taken from 33 liver grafts. Sample A was taken immediately before cold perfusion (baseline) and sample B 2 h after portal reperfusion. Donors were from one of four groups: living (LD); after cardiac death (DCD); after brain death, with subsequent post-implantation EAD in the recipient (DBD-EAD); and after brain death without EAD (DBD). RNA was extracted from the 66 samples and analyzed using Illumina Beadarray technology. A group of 3 samples from healthy volunteers and 3 samples form LD taken at the start of surgery are included as controls or reference samples. This dataset is part of the TransQST collection.

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Resolving lipids isomers up to the double bond positional level using reversed-phase chromatography, EAD fragmentation with high-resolution mass spectrometry: Application to pancreatic cancer

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