Project description:Transcriptome profiling of three models with impaired insulin/IGF1 signaling. 1. Deep sequencing of endogenous mRNA from Caenorhabditis elegans N2 var. Bristol (wildtype) and daf-2(e1370) mutant; 2. Deep sequencing of endogenous mRNA from murine embryonic fibroblasts (MEF) wildtype and irs1-/- knockout; 3. Deep sequencing of endogenous mRNA from murine embryoinic fibroblast (MEF) insr+/- -lox and insr+/- knockout 14 samples examined: C. elegans N2 var. Bristol (wildtype) vs. daf-2(e1370) mutant; MEF wildtype vs. irs1-/- knockout; MEF insr+/- -lox vs. insr +/- knockout
Project description:The major virulence factor of Plasmodium falciparum parasites, PfEMP1 is expressed by a multigene family, termed var genes. Here selection linked integration (SLI) was utilized to modify var genes in P. falciparum parasites to select for parasite populations expressing a single var gene. Bulk RNA was isolated from ring stage parasites of these SLI parasite populations and analyzed with next generation sequencing. The proportion of exon 2 transcripts of var genes normalized to transcripts per million was determined per cell line to confirm the predominant expression of the desired var gene.
Project description:We have investigated the effect of RRP6 depletion on the transcriptome of S2 cells using Illumina deep RNA sequencing. We have also carried out Illumina ChIP-seq analysis of RRP6 genome occupancy in control S2 cells (GFP-KD) and in cells depleted of SU(VAR)3-9.
Project description:Epigenetic regulation of mutually exclusive transcription within the var gene family is important for infection and pathogenesis of the malaria parasite Plasmodium falciparum. var genes are kept transcriptionally silent via heterochromatic clusters located at the nuclear periphery; however, only a few proteins have been shown to play a direct role in var gene transcriptional regulation. Importantly, the chromatin components that contribute to var gene nuclear organization remain unknown. Here, we adapted a CRISPR-based immunoprecipitation-mass spectrometry approach for de novo identification of factors associated with specific transcriptional regulatory sequences of var genes. Tagged, catalytically inactive Cas9 (“dCas9”) was targeted to var gene promoters or introns, cross-linked, and immunoprecipitated with all DNA, proteins, and RNA associated with the targeted locus. Chromatin immunoprecipitation followed by sequencing demonstrated that genome-wide dCas9 binding was specific and robust. Proteomics analysis of dCas9-immunoprecipitates identified specific proteins for each target region, including known and novel factors such as DNA binding proteins, chromatin remodelers, and structural proteins. We also demonstrate the ability to immunoprecipitate RNA that is closely associated to the targeted locus. Our CRISPR/dCas9 study establishes a new tool for targeted purification of specific genomic loci and advances understanding of virulence gene regulation in the human malaria parasite.
Project description:Transcriptome profiling of three models with impaired insulin/IGF1 signaling. 1. Deep sequencing of endogenous mRNA from Caenorhabditis elegans N2 var. Bristol (wildtype) and daf-2(e1370) mutant; 2. Deep sequencing of endogenous mRNA from murine embryonic fibroblasts (MEF) wildtype and irs1-/- knockout; 3. Deep sequencing of endogenous mRNA from murine embryoinic fibroblast (MEF) insr+/- -lox and insr+/- knockout Jena Centre for Systems Biology of Ageing - JenAge (www.jenage.de)
Project description:Oilseed mustard, Brassica juncea, exhibits high levels of genetic variability for salinity tolerance. To obtain the global view of transcriptome and investigate the molecular basis of salinity tolerance in a salt-tolerant variety CS52 of B. juncea, we performed transcriptome sequencing of control and salt-stressed seedlings. De novo assembly of 184 million high-quality paired-end reads yielded 42,327 unique transcripts longer than 300 bp with RPKM ≥1. When compared with non-redundant proteins, we could annotate 67% unigenes obtained in our study. Based on the mapping to expressed sequence tags (ESTs), 52.6% unigenes are novel compared to EST data available for B. juncea and constituent genomes. Differential expression analysis revealed altered expression of 1469 unigenes in response to salinity stress. Of these, 587, mainly associated with ROS detoxification, sulfur assimilation and calcium signaling pathways, are up regulated. Notable of these is RSA1 (SHORT ROOT IN SALT MEDIUM 1) INTERACTING TRANSCRIPTION FACTOR 1 (RITF1) homolog up regulated by >100 folds in response to stress. RITF1, encoding a bHLH transcription factor, is a positive regulator of SOS1 and several key genes involved in scavenging of salt stress-induced reactive oxygen species (ROS). Further, we performed comparative expression profiling of key genes implicated in ion homeostasis and sequestration (SOS1, SOS2, SOS3, ENH1, NHX1), calcium sensing pathway (RITF1) and ROS detoxification in contrasting cultivars, B. juncea and B. nigra, for salinity tolerance. The results revealed higher transcript accumulation of most of these genes in B. juncea var. CS52 compared to salt-sensitive cultivar even under normal growth conditions. Together, these findings reveal key pathways and signaling components that contribute to salinity tolerance in B. juncea var. CS52. We report transcriptome sequencing of two-weeks-old seedlings of B. juncea var. CS52 under normal growth conditions (CTRL) and in response to salinity stress (SS) using Illumina paired-end sequencing
Project description:Comparison of small RNA fractions derived from piRNA clusters and transposon sequences in control and Su(var)3-7 null mutant. Small RNA profile of 3-day old control and Su(var)3-7 mutant ovaries were generated by high-throughput sequencing on Illumina HiSeq 2000
Project description:We have investigated the effect of RRP6 depletion on the transcriptome of S2 cells using Affymetrix whole-genome tiling arrays. We have also carried out Illumina ChIP-seq analysis of RRP6 genome occupancy in control S2 cells (GFP-KD) and in cells depleted of SU(VAR)3-9.
Project description:Ficus deltoidea Jack (Moraceae) is a plant used in Malaysia for various conditions including as a supplement in diabetes management. Morphology distinction of the 7 varieties (var. angustifolia, var. bilobata, var. deltoidea, var. intermedia, var. kunstleri, var. motleyana and var. trengganuensis) is challenging due to the extreme leaf heterophylly and unclear varietal boundaries, making it difficult for authentication and quality control of F. deltoidea products. In this study, we applied untargeted UHPLC-HRMS metabolomics and a genetic approach to the authentication of 7 F. deltoidea varieties collected from 6 locations throughout Peninsular Malaysia. The PCA and HCA analysis showed 3 main chemotypes based on the differentiation in the main flavonoids content; var. terengganuensis and var. intermedia (chemotypes-a), var. angustifolia (chemotype-b) and var. bilobata, var. deltoidea, var. kunstleri and var. motleyana (chemotype-c). Using these groupings, the supervised PLS-DA analysis identified 15 glycosylated flavones as markers together with 1 furanocoumarin. These markers were always consistent for the respective varieties, regardless of the geographical origin and if the plants were of wild or cultivated. Additionally, the chemotaxonomy differentiation was also validated by DNA sequencing. In particular, var. bilobata accession which showed divergent morphology was also differentiated by the chemical fingerprints and genotype. Our results suggest that the chemotype differentiation based on the flavonoids fingerprints along with the proposed markers provide a complementary tool to morphology and genetic analyses for the quality control of raw materials and products from F. deltoidea varieties.
Project description:The parasite Plasmodium falciparum is responsible for severe malaria, which remains a major cause of death, particularly in sub-Saharan Africa. The reference strain NF54 (or its subclone 3D7) is commonly used for controlled human malaria infection (CHMI), but recently strains with a different geographic and genomic background have become available for CHMI, including 7G8, which was subcloned from the Brazilian isolate IMTM22 in 1984 (Burkot TR et al. 1984. Infectivity to mosquitoes of Plasmodium falciparum clones grown in vitro from the same isolate. Trans R Soc Trop Med Hyg 78 (3):339-41. doi: 10.1016/0035-9203(84)90114-7). In contrast to NF54, in which var gene expression resets after mosquito transmission, 7G8 shows a partial reset with retention of the C-type var gene PF7G8_040025600 in the human host. The three subclones A1G9 (almost exclusive var2csa expression, control), A2E10, and A2G2 (both predominantly expressing var gene PF7G8_040025600) recently obtained by limited dilution from the Sanaria 7G8 parasite working cell bank (Lot: SAN03-021214 dated 20. February 2014) were selected for gDNA sequencing to test whether parasite subclones expressing PF7G8_040025600 differ in their genomic background. 150 mL of P. falciparum cell culture with >10% parasitemia was harvested and gDNA isolation was performed using the MasterPure™ Complete DNA Purification Kit (Lucigen). The gDNA samples were tested for degradation and RNA contamination on an agarose gel and quantified using the Qubit™ dsDNA BR Assay Kit (ThermoFischer). DNA-seq was performed at BGI Genomics (Shenzhen, China) on the DNBseq platform to generate 150 bp paired-end sequencing reads.