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- DNA-seq protocols
Our collaborators and customers are continually making scientific breakthroughs. Here are the latest published results of epigenetics studies obtained using ThruPLEX DNA-seq kits for ChIP-seq and CUT&RUN studies, plus those involving small RNA sequencing using the SMARTer smRNA-Seq Kit for Illumina.
ChIP-seq (chromatin immunoprecipitation sequencing)
Baejen, C. et al. Genome-wide analysis of RNA Polymerase II termination at protein-coding. Genes. Mol. Cell 66, 1–12 (2017).
This paper used ChIP-seq, ChIP-qPCR, and other functional genomic methods to understand how RNA Pol II termination occurs in yeast. The ThruPLEX DNA-Seq Kit was used to generate libraries for ChIP-seq. The author showed that the 3′ transition in budding yeast requires the Pol II elongation factor Spt5, and that polymerase II release from DNA requires the Rat1 exonuclease. Read now »
Liu, Y. et al. Transcriptional landscape of the human cell cycle. PNAS 114, 3473–78 (2017).
This paper investigated the transcriptional landscape across the cell cycles using a combination of ChIP-seq, DNase-seq, RNA-seq, and GRO-seq. ThruPLEX DNA-Seq Kit was used to prepare libraries for ChIP-seq and DNase-seq. Using the MCF-7 breast cancer cell line as a model, the authors revealed lag between transcription and steady-state RNA expression at the cell-cycle level. Other findings highlighted the importance of transcriptional and epigenetic dynamics during cell-cycle progression. Read now »
Maatouk, D. M. et al. Genome-wide identification of regulatory elements in Sertoli cells. Development 144, 720–30 (2017).
This study used ChIP-seq, DNaseI-seq, and RNA-seq to identify regulatory elements in mouse Sertoli cells during sex determination. ThruPLEX DNA-Seq Kit was used to prepare ChIP-seq libraries from FACS-sorted mouse Sertoli cells. By overlapping DNaseI-seq peaks with the chromatin landscape for H3K27ac, the authors were able to identify enhancers active only in Sertoli cells during the early stages of sex determination. Read now »
Cejas, P. et al. Chromatin immunoprecipitation from fixed clinical tissues reveals tumor-specific enhancer profiles. Nat Med. 22, 685–691 (2016).
This paper describes a new method called fixed-tissue chromatin immunoprecipitation sequencing (FiT-seq) for reliable extraction of soluble chromatin from FFPE tissue samples for accurate detection of histone marks. ThruPLEX DNA-Seq Kit was used to generate FiT-seq libraries from FFPE specimens and ChIP-seq libraries fresh frozen samples, and data from the two sample types were demonstrated to be concordant with each other. The study shows that FiT-seq allows tumor-specific enhancers and super-enhancers to be elucidated and correlated with known oncogenic drivers to enhance understanding of how chromatin states affect gene regulation. Read now »
Hojo, H. et al. Sp7/Osterix is restricted to bone-forming vertebrates where it acts as a Dlx co-factor in osteoblast specification. Dev. Cell 37, 1–16 (2016).
This study used ChIP-seq and RNA-seq to analyze the role of the transcription factor Sp7/Osterix during bone formation in mice. The authors generated a transgenic mouse line in which a biotin motif and three FLAG epitopes were attached to the C-terminus of the Sp7 protein to enable the binding sites of Sp7 could be identified by ChIP. ThruPLEX DNA-Seq Kit was used to generate ChIP-seq libraries to identify osteoblast enhancers. The authors concluded that the appearance of Sp7 within the Sp family was likely to have played a key role in the emergence of bone-forming osteoblasts during vertebrate evolution. Read now »
Luizon, M. R. et al. Genomic characterization of metformin hepatic response. PLOS Genet. 12, e1006449 (2016).
This study identified genes and regulatory elements involved in metformin hepatic response in human hepatocytes using ChIP-seq and RNA-seq. ChIP-seq libraries were prepared with ThruPLEX DNA-Seq Kit. The paper provided a comprehensive genome-wide understanding of metformin-dependent response in human hepatocytes and identified potential candidates for the treatment of type 2 diabetes. Read now »
Roy, N. et al. PDX1 dynamically regulates pancreatic ductal adenocarcinoma initiation and maintenance. Genes Dev. 30, 2669–83 (2016).
This study used ChIP-seq, RNA-seq, and BrU-seq to uncover the diverse functions of PDX1 at different stages of pancreatic ductal adenocarcinoma (PDA). ThruPLEX DNA-Seq Kit was used to prepare ChIP-seq libraries from PDX1-immunoprecipitated DNA. The authors reported distinct roles of PDX1 at different stages of PDA, suggesting that therapeutic approaches against this potential target need to account for its changing functions at different stages of carcinogenesis. Read now »
Si, S. et al. Loss of Pcgf5 affects global H2A monoubiquitination but not the function of hematopoietic stem and progenitor cells. PLOS One 11, e0154561 (2016).
This paper analyzed the role of the Polycomb-group (PcG) RING finger protein Pcgf5 in hematopoietic stem and progenitor cells (HSPCs) using ChIP-seq and RNA-seq. Libraries for ChIP-seq were generated using ThruPLEX DNA-Seq Kit. ChIP-seq analysis confirmed the reduction in H2AK119ub1 levels observed in pcgf5-deficient HSPCs but revealed no significant association with gene expression levels. The authors concluded that Pcgf5 regulates histone H2AK119 monoubiquitination in vivo, but its role in hematopoiesis is marginal. Read now »
Spangle, J. M. et al. PI3K/AKT signaling regulates H3K4 methylation in breast cancer. Cell Rep. 15, 1–13 (2016).
Using ChIP-seq and RNA-seq, this study investigated the role of PI3K/AKT pathway activation in preclinical models of breast cancer. ChIP-seq libraries were prepared using ThruPLEX DNA-Seq Kit to measure H3K4me3 and KDM5A subcellular localization. The authors demonstrated the importance of the PI3K/AKT signaling pathway in regulating the epigenetic landscape in breast malignancies, and more specifically that the expression of cell-cycle genes regulated by the AKT/KDM5A complex is associated with advanced-stage breast cancer. Read now »
Warrick, J. I. et al. FOXA1, GATA3 and PPARγ cooperate to drive luminal subtype in bladder cancer: A molecular analysis of established human cell lines. Sci. Reps. 6, 38531 (2016).
This paper used ChIP-seq and RNA-seq to identify a set of human cell lines suitable for the study of molecular subtypes in bladder cancer. ChIP-seq libraries were prepared with ThruPLEX DNA-Seq Kit. The study shows that the combined overexpression of PPARγ, GATA3, and FOXA1 contributes to the transdifferentiation of bladder cancer cells from a more aggressive basal phenotype to a less invasive luminal phenotype. Read now »
CUT&RUN-seq (Cleavage Under Targets and Release Using Nuclease sequencing)
Ernst, C. et al. Staged developmental mapping and X chromosome transcriptional dynamics during mouse spermatogenesis. Nat. Commun. 10, 1251 (2019).
This study used CUT&RUN-seq to examine epigenetic control of the X chromosome. It focused on the inactivation and reactivation of the X chromosome following transcriptional silencing, and provides insights into the epigenetic control of X chromosome reactivation in post-meiotic spermatids. Sequencing libraries were prepared using the ThruPLEX DNA-Seq Kit with some modifications. Read now »
Stojic, L. et al. A high-content RNAi screen reveals multiple roles for long noncoding RNAs in cell division. bioRxiv 709030 (2019). doi: https://doi.org/10.1101/709030
The aim of this study was to discover and elucidate the role of lncRNA in cell division, using a high-content RNAi screening method that involved depletion of 2,231 lncRNAs in HeLa cells. Selected lncRNAs were then used for genome-wide profiling of active and repressive histone modifications as well as transcription factors using the CUT&RUN technique. The lncRNA binding sites on chromatin were identified using capture hybridization analysis of RNA targets with sequencing (CHART-seq). The ThruPLEX DNA-Seq Kit was used to prepare both CUT&RUN-seq and CHART-seq libraries for this study. Read now »
smRNA-seq (small RNA sequencing)
Dadonaite, B., Barilaite, E., Fodor, E., Laederach, A. & Bauer, D. L. The structure of the influenza A virus genome. bioRxiv 236620 (2017). doi:10.1101/236620
In this study, the researchers utilized the SMARTer smRNA-Seq Kit for Illumina to investigate RNA-RNA interactions inside influenza virions. This study provides details on how influenza virus controls its packaging and growth, as well as the mechanism of potential reassortment of genomes among different strains. The article discusses how this data can be used to predict the emergence of new pandemic influenza strains. Read now »
Griswold, A. J. et al. Transcriptomic analysis of synovial extracellular RNA following knee trauma: A pilot study. J. Orthop. Res. (2017). doi:10.1002/jor.23802
Extracellular RNA (exRNA) from synovial fluids of post-traumatic knee injury patients was profiled using the SMARTer smRNA-Seq Kit for Illumina. The study identified several matrix metalloproteinases by analyzing exRNA fragments in synovial fluid samples obtained from patients. Read now »
Pasquariello, R. et al. Micro-RNA sequencing of individual human oocytes. Fertil. Steril. 108, e144 (2017).
In this study, the researchers performed miRNA profiling on human MII oocytes using the SMARTer smRNA-Seq Kit for Illumina. The article discusses a novel methodology for profiling individual oocytes and suggests an application using miRNAs as markers for reproductive failure. Read now »
Hornstein, N. et al. Ligation-free ribosome profiling of cell type-specific translation in the brain. Genome Biol. 17, 149 (2016).
In this study, the authors developed a method for ligation-free ribosomal profiling using the SMARTer smRNA-Seq Kit for Illumina. They used this approach with mouse brain tissue to identify targets of mTOR signaling in the brain. Read now »
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