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A novel mammalian gene controls mammal-specific neuronal functioning [ChIP-Exo]

ABSTRACT: In mammals brain evolution, specifically that of the cerebellum, is a focus of evolutionary change. Here we functionally characterize the phylogenetically-restricted novel gene, piggyBac transposable element. The ChiP-exonuclease assay protocol was performed as in Serandour's method. The libraries were quantified by using the KAPA library quantification kit for Illumina sequencing platforms (KAPA Biosystems, KK4824) and sequenced on HiSeq following the manufacturer’s protocol.

ORGANISM(S): Homo sapiens

PROVIDER: GSE213758 | GEO | 2022/09/23

REPOSITORIES: GEO

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A novel mammalian gene controls mammal-specific neuronal functioning [RNA-Seq]

Project description:n mammals brain evolution, specifically that of the cerebellum, is a focus of evolutionary change. Here we functionally characterize the phylogenetically-restricted novel gene, piggyBac transposable

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A novel mammalian gene controls mammal-specific neuronal functioning [ChIP-Exo]

Project description:A novel mammalian gene controls mammal-specific neuronal functioning [ChIP-Exo]

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Project description:To identify the expression profiles of circRNAs in peripheral blood mononuclear cells (PBMCs) from Ankylosing Spondylitis (AS) patients and healthy controls, we used circRNA sequencing to detect circRNA in 3 samples of PBMCs from AS patients and 3 samples of PBMCs from healthy controls. In brief, total RNA was extracted and purified using Magen Hipure Total RNA Mini Kit (Magen, Guangzhou, China), followed by constructing RNA sequencing libraries with KAPA RNA HyperPrep Kit with RiboErase (Kapa Biosystems, Inc., MA, USA). After the construction of RNA sequencing libraries, Qubit 3.0 fluorometer (Invitrogen, CA, USA) and the Agilent 2100 bioanalyzer (Applied Biosystems, CA, USA) were used for quality control. Then, the PE150 mode of HiSeq X10 (Illumina Inc., CA, USA) was used for sequencing. Differentially expressed circRNAs were screened with criteria of fold-change>1.5 and p<0.05.

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Project description:Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) and its ultra-high resolution cousin ChIP-exo are methods that identify where proteins bind along any genome in vivo. ChIP-exo achieves near-base pair resolution by creating exonuclease stop sites just 5’ to where formaldehyde-induced protein-DNA cross-links occur. Whereas construction of ChIP genomic libraries is straightforward and widely adopted for ChIP-seq, ChIP-exo is technically more involved which has resulted in limited adoption. Here we describe multiple ChIP-exo protocols, each with use-specific advantages and limitations. The new versions are greatly simplified through removal of multiple enzymatic steps. This is achieved in part through the use of Tn5 tagmentation and/or single-stranded DNA ligation. The result is greater library yields, lower processing time, and lower cost. A similar streamlined approach was developed for ChIP-seq, called ChIP-seq 1-step, where library construction is achieved in one-step.

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ChIP-nexus: a novel ChIP-exo protocol for improved detection of in vivo transcription factor binding footprints

Project description:Understanding how eukaryotic enhancers are bound and regulated by specific combinations of transcription factors is still a major challenge. To better map transcription factor binding genome-wide at nucleotide resolution in vivo, we have developed a robust ChIP-exo protocol called ChIP-nexus (chromatin immunoprecipitation experiments with nucleotide resolution through exonuclease, unique barcode and single ligation), which utilizes an efficient DNA self-circularization step during library preparation. Application of ChIP-nexus to four proteins—human TBP, Drosophila NFkB, Twist and Max—showed that it outperformed existing ChIP protocols in resolution and specificity, pinpointed relevant binding sites within enhancers containing multiple binding motifs, and allowed for the analysis of in vivo binding specificities. Notably, we show that Max frequently interacted with DNA sequences next to its motif, and that this binding pattern correlated with local DNA-sequence features such as DNA shape. ChIP-nexus will be broadly applicable to the study of in vivo transcription factor binding specificity and its relationship to cis-regulatory changes in humans and model organisms.

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