A 3’-end capture sequencing method for high-throughput targeted gene expression profiling
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE190096 | GEO | 2021/12/05
REPOSITORIES: GEO
Similar Datasets
Project description:A 3’-end capture sequencing method for high-throughput targeted gene expression profiling
| PRJNA785830 | ENA
Project description:BackgroundSignificant efforts have recently been put into the investigation of the spatial organization and the chromatin-interaction networks of genomes. Chromosome conformation capture (3C) technology and its derivatives are important tools used in this effort. However, many of these have limitations, such as being limited to one viewpoint, expensive with moderate to low resolution, and/or requiring a large sequencing effort. Techniques like Hi-C provide a genome-wide analysis. However, it requires massive sequencing effort with considerable costs. Here we describe a new technique termed Targeted Chromatin Capture (T2C), to interrogate large selected regions of the genome. T2C provides an unbiased view of the spatial organization of selected loci at superior resolution (single restriction fragment resolution, from 2 to 6 kbp) at much lower costs than Hi-C due to the lower sequencing effort.ResultsWe applied T2C on well-known model regions, the mouse β-globin locus and the human H19/IGF2 locus. In both cases we identified all known chromatin interactions. Furthermore, we compared the human H19/IGF2 locus data obtained from different chromatin conformation capturing methods with T2C data. We observed the same compartmentalization of the locus, but at a much higher resolution (single restriction fragments vs. the common 40 kbp bins) and higher coverage. Moreover, we compared the β-globin locus in two different biological samples (mouse primary erythroid cells and mouse fetal brain), where it is either actively transcribed or not, to identify possible transcriptional dependent interactions. We identified the known interactions in the β-globin locus and the same topological domains in both mouse primary erythroid cells and in mouse fetal brain with the latter having fewer interactions probably due to the inactivity of the locus. Furthermore, we show that interactions due to the important chromatin proteins, Ldb1 and Ctcf, in both tissues can be analyzed easily to reveal their role on transcriptional interactions and genome folding.ConclusionsT2C is an efficient, easy, and affordable with high (restriction fragment) resolution tool to address both genome compartmentalization and chromatin-interaction networks for specific genomic regions at high resolution for both clinical and non-clinical research.
| S-EPMC4100494 | biostudies-literature
Project description:Long-read RNA sequencing (RNA-seq) is a powerful technology for transcriptome analysis, but the relatively low throughput of current long-read sequencing platforms limits transcript coverage. We present TEQUILA-seq, a versatile, easy-to-implement, and low-cost method for targeted long-read RNA-seq. TEQUILA-seq can be broadly used for targeted sequencing of full-length transcripts in diverse biomedical research settings.
2023-06-06 | GSE213984 | GEO
Project description:We describe a differential cellular lysis strategy, followed by a biotynilated probe-based ribosomal depletion, to simultaneously obtain the gene expression of Mycobacterium tuberculosis infecting BALB/c mouse lungs
2021-01-27 | GSE160357 | GEO
Project description:Chromosomal translocations require formation and joining of DNA double strand breaks (DSBs). These events disrupt the integrity of the genome and are involved in producing leukemias, lymphomas and sarcomas. Translocations are frequent, clonal and recurrent in mature B cell lymphomas, which bear a particularly high DNA damage burden by virtue of activation-induced cytidine deaminase (AID) expression. Despite the ubiquity of genomic rearrangements, the forces that underlie their genesis are not well understood. Here, we provide a detailed description of a new method for studying these events, translocation capture sequencing (TC-Seq). TC-Seq provides the means to document chromosomal rearrangements genome-wide in primary cells, and to discover recombination hotspots. Demonstrating its effectiveness, we successfully estimate the frequency of c-myc/IgH translocations in primary B cells, and identify hotspots of AID-mediated recombination. Furthermore, TC-Seq can be adapted to generate genome-wide rearrangement maps in any cell type and under any condition.
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Project description:Next-generation sequencing (NGS) allows faster acquisition of metagenomic data, but complete exploration of complex ecosystems is hindered by the extraordinary diversity of microorganisms. To reduce the environmental complexity, we created an innovative solution hybrid selection (SHS) method that is combined with NGS to characterize large DNA fragments harbouring biomarkers of interest. The quality of enrichment was evaluated after fragments containing the methyl coenzyme M reductase subunit A gene (mcrA), the biomarker of methanogenesis, were captured from a Methanosarcina strain and a metagenomic sample from a meromictic lake. The methanogen diversity was compared with direct metagenome and mcrA-based amplicon pyrosequencing strategies. The SHS approach resulted in the capture of DNA fragments up to 2.5 kb with an enrichment efficiency between 41 and 100%, depending on the sample complexity. Compared with direct metagenome and amplicons sequencing, SHS detected broader mcrA diversity, and it allowed efficient sampling of the rare biosphere and unknown sequences. In contrast to amplicon-based strategies, SHS is less biased and GC independent, and it recovered complete biomarker sequences in addition to conserved regions. Because this method can also isolate the regions flanking the target sequences, it could facilitate operon reconstructions.
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Project description:Targeted paired-end sequencing of cDNA from unfragmented nascent RNA from exponentially growing S. cerevisiae cells was employed to obtain Pol II transcription elongation and splicing information from single transcripts. Nascent RNA was prepared from the yeast chromatin fraction (Carrillo Oesterreich, Preibisch, Neugebauer, Mol Cell 2010) or enriched from total RNA with polyadenylated RNA depletion. The nascent 3â end was labeled with a 3â DNA adaptor through ligation. A PCR with a forward primer in the first exon of select intron-containing genes amplifies nascent transcripts of specific genes and ensures sequencing adaptor attachment for paired-end sequencing. With this approach co-transcriptional splicing progression with distance from the intron end could be analyzed for 87 genes. Note that the unmapped and mapped data also include genes that did not pass the read coverage requirements in SMIT analysis. Nascent RNA profiles for mainly intron-containing genes were generated with paired-end sequencing with Illumina HiSeq technology.
2016-03-24 | E-GEOD-70907 | biostudies-arrayexpress
Project description:We generated a genome wide map of instances where the long noncoding RNA, Tug1, binds to DNA in cultured mouse podocytes under normal glucose conditions using Chromatin-RNA Precipitation coupled with high throughput sequencing (ChIRP-Seq) 48 alternating (even, odd) biotynilated probes were designed to span the full length of Tug1 RNA. Chromatin was prepared from gluteraldehyde crosslinked nuclei from early passage podocytes. Chromatin extracts were duplicated with either even or odd probes. Duplicate samples for Input DNA, Even pulldown (PD) and Odd PD DNA was purified following incubation and supplied for Illumina sequencing by ArrayStar (Rockville, MD).
2016-10-24 | E-GEOD-77493 | biostudies-arrayexpress
Project description:Targeted paired-end sequencing of cDNA from unfragmented nascent RNA from exponentially growing S. cerevisiae cells was employed to obtain Pol II transcription elongation and splicing information from single transcripts. Nascent RNA was prepared from the yeast chromatin fraction (Carrillo Oesterreich, Preibisch, Neugebauer, Mol Cell 2010) or enriched from total RNA with polyadenylated RNA depletion. The nascent 3’ end was labeled with a 3’ DNA adaptor through ligation. A PCR with a forward primer in the first exon of select intron-containing genes amplifies nascent transcripts of specific genes and ensures sequencing adaptor attachment for paired-end sequencing. With this approach co-transcriptional splicing progression with distance from the intron end could be analyzed for 87 genes. Note that the unmapped and mapped data also include genes that did not pass the read coverage requirements in SMIT analysis.
2016-03-24 | GSE70907 | GEO
Project description:Targeted gene insertion is a goal of genome editing and has been performed in cultured cells but only in a handful of whole organisms. The existing method to integrate foreign DNA using the homologous recombination pathway is inherently low efficiency, and many systems are refractory to this method. Several additional manipulations have been developed to gain greater efficiency by suppressing the competing dominant repair pathway of nonhomologous end-joining. However, this can be laborious and in practice limits the range of hosts where the method is applicable. Here, we use the preferred pathway of nonhomologous end-joining (used previously to create indels for gene inactivation) for precise integration of large DNA into the specified genomic target site of an intact animal. Our method uses site-specific cleavage, end-capture of cohesive ends, and obligate ligation-gated recombination. This approach is straight-forward and yields high efficiency without additional gene manipulations; therefore it is easily applicable to a much broader range of organisms. We demonstrate its application to the fungus fly Sciara coprophila where a transformation system has not existed before. We integrated a 6.5 kb transgene precisely at the desired genomic target site of Sciara using this method. This provides the foundation for future experiments to explore the unique genetic features of this organism. Similarly, the method described here will allow insertion of large pieces of DNA into a diverse group of organisms for studies of their genetic attributes.
| S-EPMC4555220 | biostudies-literature