Dataset Information

RAD18_RAD6_MAGEA4_Cross-linking_Mass_Spectrometry

ABSTRACT: RAD18 and RAD6 were co-expressed in E.coli, RAD18 harbors an N-terminal 6His tag which was utilized for affinity chromatography via cobalt affinity resin. The complex was then subjected to size exclusion chromatography. 5 X molar excess MAGEA4 (also has N-terminal His-tag) was added to RAD18-RAD6. The complex was crosslinked with 0.5 x Molar BS3 to lysine residues within the complex for 10 minutes at room temperature, shaking at 600 rpm. The reaction was quenched with 100 mM Tris pH 8, incubated at 35 Â°C for 10 minutes, shaking at 600 rpm.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Frank Stein

LAB HEAD: Sagar Bhogaraju

PROVIDER: PXD047929 | Pride | 2024-02-20

REPOSITORIES: Pride

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Project description:Since their discovery, transposable elements have been proposed to play a central role in the evolution of their host genomes through their ability to regulate gene expression, in particular by providing transcription start sites (TSSs) for host genes. To investigate their contribution to developmental gene expression, we developed RAMPAGE, a high-throughput 5'-complete cDNA sequencing approach to accurately discover TSSs, characterize their transcripts, and quantify their expression. This strategy, which directly delineates the expression profiles of individual promoters and was designed to offer optimal sample multiplexing capabilities, represents an advantageous alternative to standard RNA-Seq for a wide range of transcriptome profiling applications. We used RAMPAGE in a genome-wide study of promoter activity throughout 36 stages of the life cycle of Drosophila melanogaster, and describe here a comprehensive dataset that represents the first developmental timecourse of promoter usage. We found that over 40% of developmentally expressed genes have at least 2 promoters, and that alternative promoters generally implement distinct regulatory programs. Transposons harbor TSSs driving the expression of hundreds of annotated genes, and they often impart their own expression specificity upon the genes they regulate. Detailed analysis of particular transposons identified sequence elements encoding these regulatory properties. Our results show that transposable elements contribute significantly to the generation of standing variation and to the evolution of gene regulatory networks, by distributing stereotyped regulatory modules throughout the genome. This dataset represents a whole-genome, single-base resolution profiling of transcription start site (TSS) expression throughout 36 stages of the life cycle of Drosophila melanogaster. These profiles were established using RAMPAGE, a high-throughput, high-accuracy 5'-complete cDNA sequencing method implemented on the Illumina platform. Embryos, larvae, pupae and adult flies were collected at specific stages of development, and RAMPAGE profiles were established for pools of whole organisms. The data was analyzed using custom scripts and algorithms that are all available upon request. Supplementary files: Dmel_Combined_+.bw: bigWig coverage by cDNA 5' ends (+ strand). Dmel_Combined_-.bw: bigWig coverage by cDNA 5' ends (- strand). Dmel_All_RAMPAGE_peaks.bed: BED file describing all RAMPAGE peaks. Dmel_GeneTSS_RAMPAGE_peaks.bed: BED file describing all peaks attributed to annotated genes. GeneTSS_expression_RAMPAGE_RPM.txt: Expression matrix for all genic peaks (RPM: reads per million). Transposon_expression_RAMPAGE_RPM.txt: Expression matrix for all RepeatMasker-annotated transposon classes (RPM: reads per million). Genome build: dm3

Dataset Information

RAD18_RAD6_MAGEA4_Cross-linking_Mass_Spectrometry

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