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Project description:Pancreatic cancer is a complex disease with a desmoplastic stroma, extreme hypoxia, and inherent resistance to therapy. Understanding the signaling and adaptive response of such an aggressive cancer is key to making advances in therapeutic efficacy and understanding disease progression. Redox factor-1 (Ref-1), a redox signaling protein, regulates the DNA binding activity of several transcription factors, including HIF-1. The conversion of HIF-1 from an oxidized to reduced state leads to enhancement of its DNA binding. In our previously published work, knockdown of Ref-1 under normoxia resulted in altered gene expression patterns on pathways including EIF2, protein kinase A, and mTOR. In this study, single cell RNA sequencing (scRNA-seq) and proteomics were used to explore the effects of Ref-1 on metabolic pathways under hypoxia.Results: We also integrated the scRNA data analysis with the proteomic analysis and found that the differentially expressed genes and pathways identified from the scRNA-seq data are highly consistent to the significant proteins observed in the proteomics data, especially for the upregulated cell cycle and transcription pathways and downregulated metabolic, apoptosis and signaling pathways under hypoxia. Conclusion: The scRNA-seq and proteomics data consistently demonstrated down-regulated central metabolism pathways in APE1/Ref-1 knockdown vs scrambled control under both normoxia and hypoxia conditions. Experimental Methods: scRNA-seq comparing pancreatic cancer cells expressing less than 20% of the Ref-1 protein was analyzed using left truncated mixture Gaussian model. Matched samples were also collected for bulk proteomic analysis of the four conditions. scRNA-seq data was validated using proteomics and qRT-PCR. Ref-1’s role in mitochondrial function was confirmed using mitochondrial function assays and qRT-PCR. Results: We also integrated the scRNA data analysis with the proteomic analysis and found that the differentially expressed genes and pathways identified from the scRNA-seq data are highly consistent to the significant proteins observed in the proteomics data, especially for the upregulated cell cycle and transcription pathways and downregulated metabolic, apoptosis and signaling pathways under hypoxia. Conclusion: The scRNA-seq and proteomics data consistently demonstrated down-regulated central metabolism pathways in APE1/Ref-1 knockdown vs scrambled control under both normoxia and hypoxia conditions.
2021-09-09 | PXD020515 | Pride
Project description:Bulked segregant analysis (BSA) is an efficient method to rapidly and efficiently map genes responsible for mutant phenotypes. This procedure, however, requires access to quantitative genetic markers that are polymorphic in the mapping population. We have developed a modification of BSA (BSR-Seq) that makes use of RNA-Seq reads to efficiently map genes even in populations for which no polymorphic markers have been previously identified. Because of the digital nature of next-generation sequencing (NGS) data, it is possible to conduct de novo SNP discovery and quantitatively genotype BSA samples using the same RNA-Seq data. In addition, analysis of the RNA-Seq data provides information on the effects of the mutant on global patterns of gene expression at no extra cost. In combination these results greatly simplify gene cloning experiments. To demonstrate the utility of this strategy BSR-Seq was used to clone the glossy3 (gl3) gene of maize. Mutants of the glossy loci exhibit altered accumulation of epicuticular waxes on juvenile leaves. We previously generated a large collection of glossy mutants using the Mu transposon system. By subjected a reference allele to BSR-Seq, we were able to map the gl3 locus to a ~2.3Mb interval that is consistent with the results of prior mapping experiments. The single gene located in the 2.3Mb mapping interval that contained a Mu insertion and whose expression was down-regulated in the mutant pool was subsequently demonstrated to be the gl3 gene via the analysis of multiple independently Mu transposon induced mutant alleles. The gl3 gene encodes a putative myb transcription factor, which directly or indirectly affects the expression of a number of genes involved in the biosynthesis of very-long-chain fatty acids.
2012-12-03 | E-MTAB-4234 | biostudies-arrayexpress
Project description:The goals of this study are to compare NGS-derived transcriptomes (RNA-Seq) derived from ded1-ref mutant and normal maize endosperm tissue. Homozygous ded1-ref mutants exhibit a seed defect. Maize Ded1 encodes a transcription factor. This study characterizes DEGs between ded1-ref and normal endosperm and serves to identify direct target genes together with DAP-seq data.
2022-06-10 | GSE183302 | GEO
Project description:N6-methyladenosine (m6A) is one of the most abundant mRNA modifications in eukaryotes, related to pivotal RNA metabolism processes. The most popular high-throughput m6A identification method relies on the commercial m6A antibody but suffers from poor reproducibility and limited resolution. Exact location of m6A site is of great vital for understanding the dynamics, functions and machinery of RNA methylation. Here, we developed a precise and high-throughput antibody-independent m6A identification method based on the m6A-sensitive RNA endoribonuclease recognizing ACA motif (m6A-sensitive RNA-Endoribonuclease–Facilitated sequencing or m6A-REF-seq). Whole-transcriptomic single base m6A map generated by m6A-REF-seq displayed a typical distribution pattern with enrichment adjacent to stop codon. Ligase-based and qPCR validation methods were used to confirm the individual m6A sites and quantify the methylation level, reinforcing the high accuracy of m6A-REF-seq. We applied m6A-REF-seq on five tissues from three mammals, showing that m6A sites were conserved and tend to gather together among species. (m6A-REF-seq had been named as Aim-seq.)
2019-07-03 | GSE125240 | GEO
Project description:Sample groupings for this project are as follows: Cauline leaf vs Cultured Cell : - Cauline leaf, p1; Exp:Red; Ref: Green (Cy5:Cy3) - Cauline leaf, p2; Exp:Red; Ref: Green (Cy5:Cy3) - Cauline leaf, p3; Exp:Red; Ref: Green (Cy5:Cy3) Cotyledon light vs Cotyledon dark : - Cotyledon, CP1; Exp:Red; Ref: Green (Cy5:Cy3) - Cotyledon, CP2; Exp:Red; Ref: Green (Cy5:Cy3) Cotyledon light vs Cultured cell : - Cotyledon, cn1; Exp:Green; Ref: Red (Cy3:Cy5) - Cotyledon, cn2; Exp:Green; Ref: Red (Cy3:Cy5) Cotyledon dark vs Cultured cell : - Cotyledon dark, n1; Exp:Green; Ref: Red (Cy3:Cy5) - Cotyledon dark, n2; Exp:Green; Ref: Red (Cy3:Cy5) - Cotyledon dark, n3; Exp:Green; Ref: Red (Cy3:Cy5) Germinating seed vs Cultured cell : - Germinating seed, gn1; Exp:Green; Ref: Red (Cy3:Cy5) - Germinating seed, gn2; Exp:Green; Ref: Red (Cy3:Cy5) - Germinating seed, gn3; Exp:Green; Ref: Red (Cy3:Cy5) Hypocotyl light vs Hypocotyl dark : - Hypocotyl, hldn1; Exp:Red; Ref: Green (Cy5:Cy3) - Hypocotyl, hldn2; Exp:Red; Ref: Green (Cy5:Cy3) Hypocotyl light vs Cultured cell : - Hypocotyl light, hln1; Exp:Green; Ref: Red (Cy3:Cy5) - Hypocotyl light, hln2; Exp:Green; Ref: Red (Cy3:Cy5) Petal vs Cultured cell : - Petal, pn1; Exp:Green; Ref: Red (Cy3:Cy5) - Petal, pn2; Exp:Green; Ref: Red (Cy3:Cy5) - Petal, pn3; Exp:Green; Ref: Red (Cy3:Cy5) 1DBP vs Cultured cell : - Pistil 1DBP, pin1; Exp:Green; Ref: Red (Cy3:Cy5) - Pistil 1DBP, pin2; Exp:Green; Ref: Red (Cy3:Cy5) - Pistil 1DBP, pin3; Exp:Green; Ref: Red (Cy3:Cy5) Pistil 1DPP vs Cultured cell : - Pistil 1DPP, psn1; Exp:Green; Ref: Red (Cy3:Cy5) - Pistil 1DPP, psn2; Exp:Green; Ref: Red (Cy3:Cy5) - Pistil 1DPP, psn3; Exp:Green; Ref: Red (Cy3:Cy5) - Pistil 1DPP, psn4; Exp:Green; Ref: Red (Cy3:Cy5) Root dark vs Cultured cell : - Root dark, rdn1; Exp:Green; Ref: Red (Cy3:Cy5) - Root dark, rdn2; Exp:Green; Ref: Red (Cy3:Cy5) Root light vs Cultured cell : - Root light, rln1; Exp:Green; Ref: Red (Cy3:Cy5) - Root light, rlp1; Exp:Red; Ref: Green (Cy5:Cy3) Root light vs Root dark : - Root light vs dark, rldp1; Exp:Red; Ref: Green (Cy5:Cy3) - Root light vs dark, rldp2; Exp:Red; Ref: Green (Cy5:Cy3) Rosette leaf vs Cultured cell : - Rosette leaf, rfp1; Exp:Red; Ref: Green (Cy5:Cy3) - Rosette leaf, rfp2; Exp:Red; Ref: Green (Cy5:Cy3) - Rosette leaf, rfp3; Exp:Red; Ref: Green (Cy5:Cy3) Sepal vs Cultured cell : - Sepal, sn1; Exp:Green; Ref: Red (Cy3:Cy5) - Sepal, sn2; Exp:Green; Ref: Red (Cy3:Cy5) Silique 3DPP vs Cultured cell : - Silique 3DPP, s3n1; Exp:Green; Ref: Red (Cy3:Cy5) - Silique 3DPP, s3n2; Exp:Green; Ref: Red (Cy3:Cy5) - Silique 3DPP, s3n3; Exp:Green; Ref: Red (Cy3:Cy5) Silique 8DPP vs Cultured cell : - Silique 8DPP, s8n1; Exp:Green; Ref: Red (Cy3:Cy5) - Silique 8DPP, s8n2; Exp:Green; Ref: Red (Cy3:Cy5) - Silique 8DPP, s8n3; Exp:Green; Ref: Red (Cy3:Cy5) Stamen vs Cultured cell : - Stamen, smn1; Exp:Green; Ref: Red (Cy3:Cy5) - Stamen, smn2; Exp:Green; Ref: Red (Cy3:Cy5) - Stamen, smn3; Exp:Green; Ref: Red (Cy3:Cy5) Hypocotyl dark vs Cultured cell : - Hypocotyl dark, hn1; Exp:Green; Ref: Red (Cy3:Cy5) - Hypocotyl dark, hn2; Exp:Green; Ref: Red (Cy3:Cy5) Stem vs Cultured cell : - Stem, stp1; Exp:Red; Ref: Green (Cy5:Cy3) - Stem, stp2; Exp:Red; Ref: Green (Cy5:Cy3) - Stem, stp3; Exp:Red; Ref: Green (Cy5:Cy3) Keywords: other
2004-10-24 | GSE1599 | GEO
Project description:To investigate the mRNA m6A modification profiling in Mesocricetus auratus cells that infected with Senecavirus, we used passage-5 Senecavirus A to infect BSR-T7/5 cells. We then performed m6A MeRIP-seq(GenSeq®️ m6A MeRIP Kit) at at two time points (12hrs or 72 hrs after infection), each time point with three replicates.
2022-06-30 | GSE206974 | GEO
Project description:Mouse primary hepatocytes (MPH) prepared from C57Bl6 male adult mice were treated with the ERS-drug thapsigargin and ChIP-seq was performed using an H3K27ac antibody (Active Motif ref 39685).
2020-04-29 | GSE122613 | GEO