Library-10000 Sequencing
Ontology highlight
ABSTRACT:
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR029955.fastq.gz | Fastqsanger.gz |
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Project description:Next-generation sequencing has been widely used for the genome-wide profiling of histone modifications, transcription factor binding and gene expression through chromatin immunoprecipitated DNA sequencing (ChIP-seq) and cDNA sequencing (RNA-seq). Here, we describe a versatile library construction method that can be applied to both ChIP-seq and RNA-seq on the widely used Illumina platforms. Standard methods for ChIP-seq library construction require nanograms of starting DNA, substantially limiting its application to rare cell types or limited clinical samples. By minimizing the DNA purification steps that cause major sample loss, our method achieved a high sensitivity in ChIP-seq library preparation. Using this method, we achieved the following: (1) generated high-quality epigenomic and transcription factor-binding maps using ChIP-seq for murine adipocytes; (2) successfully prepared a ChIP-seq library from as little as 25 pg of starting DNA; (3) achieved paired-end sequencing of the ChIP-seq libraries; (4) systematically profiled gene expression dynamics during murine adipogenesis using RNA-seq; and (5) preserved the strand specificity of the transcripts in RNA-seq. Given its sensitivity and versatility in both double-stranded and single-stranded DNA library construction, this method has wide applications in genomic, epigenomic, transcriptomic and interactomic studies. Pre-adipocytes and mature adipocytes were collected. Their chromatin and RNA were subjected to ChIP and mRNA extraction. Sequencing libraries from ChIP DNA or mRNA were generated following either standard protocols or TELP method. The quality and features of TELP libraries were proved and demonstrated in comparison with standard libraries or other published data.
2015-12-15 | E-GEOD-75966 | biostudies-arrayexpress
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2011-11-02 | E-GEOD-33380 | biostudies-arrayexpress
Project description:The ligation step in RNA sequencing library generation is a known source of bias. We present the first comparison of the standard duplex adaptor protocol supplied by Life Technologies for use on the Ion Torrent PGM with an alternate single adaptor approach involving CircLigase (CircLig). We also investigate whether using the thermostable ligase Methanobacterium thermoautotrophicum RNA ligase K97A (Mth K97A) for the initial ligation step in the CircLigase protocol reduces bias. A pool of small RNA fragments of known composition was converted into a sequencing library using one of three protocols and sequenced on an Ion Torrent PGM. The single adaptor CircLigase-based approach significantly reduces, but does not eliminate, bias in Ion Torrent data. Using Mth K97A as part of the CircLig method does not further reduce bias.
2014-06-24 | E-MTAB-2566 | biostudies-arrayexpress
Project description:Next-generation sequencing has been widely used for the genome-wide profiling of histone modifications, transcription factor binding and gene expression through chromatin immunoprecipitated DNA sequencing (ChIP-seq) and cDNA sequencing (RNA-seq). Here, we describe a versatile library construction method that can be applied to both ChIP-seq and RNA-seq on the widely used Illumina platforms. Standard methods for ChIP-seq library construction require nanograms of starting DNA, substantially limiting its application to rare cell types or limited clinical samples. By minimizing the DNA purification steps that cause major sample loss, our method achieved a high sensitivity in ChIP-seq library preparation. Using this method, we achieved the following: (1) generated high-quality epigenomic and transcription factor-binding maps using ChIP-seq for murine adipocytes; (2) successfully prepared a ChIP-seq library from as little as 25 pg of starting DNA; (3) achieved paired-end sequencing of the ChIP-seq libraries; (4) systematically profiled gene expression dynamics during murine adipogenesis using RNA-seq; and (5) preserved the strand specificity of the transcripts in RNA-seq. Given its sensitivity and versatility in both double-stranded and single-stranded DNA library construction, this method has wide applications in genomic, epigenomic, transcriptomic and interactomic studies.
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Project description:Advances in Next Generation Sequencing (NGS) have made available a wealth of information that had previously been inaccessible to researchers and clinicians. NGS has been applied to understand genomic, transcriptomic, and epigenomic changes and gained traction as a significant tool capable of accelerating diagnosis, prognosis, and biomarker discovery. However, these NGS assays have yet to be practical methods for patient stratification or diagnosis because of the gap between the tiny quantities of biomaterials provided by a clinical sample and the large DNA input required by most of these assays. Current library preparation methodologies typically require large input amounts of DNA and a long and complicated manual process. Here we present a microfluidic reactor system for NGS library preparation, capable of reducing the number of pipetting steps significantly, automating much of the process, while supporting extremely low DNA input requirement (10 pg per library). This largely automated technology will allow for low-input preparations of 8 libraries simultaneously while reducing batch to batch variation and operator hands-on time.
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Project description:We developed a high-throughput mutagenesis screen to comprehensively identify the cis-regulatory elements that control a target splicing event from the MST1R gene that codes for the RON receptor tyrosine kinase. Skipping of alternative exon 11 results in a constitutively active isoform that promotes epithelial to mesenchymal transition and thereby contributes to the invasive phenotype of tumors. First, we created a library of mutated minigenes via mutagenic PCR. Importantly, the reverse primer introduced a random barcode sequence which labels the associated mutations. Next, the plasmid library was transfected as a pool and depending on the mutations, the transcripts exhibit changes in alternative splicing. The minigene library and the splicing outcome were analyzed by next-generation sequencing and subsequent integration of the datasets resulted in a map of splicing regulatory sites. The DNA-seq experiment was performed to map the mutations and the associated barcodes in order to identify all minigene variants in the library. For sequencing, we generated five overlapping amplicons of the minigene library using four different forward primers: 5’CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNNNNNNCTATAGGGAGACCCAAGCTT 3’, 5’CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNNNNNNGTTCCACTGAAGCCTGAG 3’, 5’CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNNNNNNAGCTGCCAGCACGAGTTC 3’, 5’CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNNNNNNGAATCTGAGTGCCCGAGG 3’, and 5’CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNNNNNNctactggctggtcctcatga 3’, and 5’AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNATAGAATAGGGCCCTCTAGA 3’ as a common reverse primer. After amplification, the PCR products were cleaned using the GeneRead size selection Kit (QIAGEN) according to manufacturer’s instructions. The purified products were first analysed with the TapeStation 2200 capillary gel electrophoresis instrument (Agilent) and then fluorimetrically quantified using a Qubit fluorimeter (Thermo Scientific). Sequencing was carried out on the Illumina MiSeq platform using paired-end reads of 300 nt length and a 10% PhiX spike-in to increase sequence complexity.
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