Project description:To find downstream target of SOC1, we attemted global expression profiles in gain-of-function mutants of SOC1; To find downstream target of SOC1, we attemted global expression profiles in soc1-101D: ; Expt. 1 GSM73643, GSM73646; Expt. 2 GSM73647, GSM73648; Expt. 3 GSM73649, GSM73650; To find downstream target of SOC1, we attemted global expression profiles in soc1-2: GSM73649, GSM73651; To find downstream target of SOC1, we attemted global expression profiles in gain-of-function mutants of SOC1: ; GSM73643, GSM73646, GSM73647, GSM73648, GSM73649, GSM73650 Experiment Overall Design: To more completely identify the downstream targets of SOC1, we attemted to compare the expression profiles of g-o-f mutant of SOC1 with the results of soc1-2 mutant
Project description:Comparison between transcriptomes of dash mutants vs WT plants and ectopic expression of 35S::DASH in hairy roots versus empty vector Loss of function: 3 WT plants A17 at 8 days after pollination and 3 dash mutant plants at 8 days afer pollination. Gain of function transformants: 3 transformed plants containing empty vector and 4 transformed plants containing 35S::DASH Total RNA was extracted using a modified cetyltrimethylammonium bromide method (Verdier et al., 2008). 5ug of total RNA from each sample was purified (RNeasy MinElute Cleanup kit; Qiagen) according to the manufacturer?s instructions. RNA was quantified and evaluated for purity using an ND-1000 Nanodrop Spectrophotometer (NanoDrop Technologies) and a Bioanalyzer 2100 (Agilent). The Affymetrix M. truncatula GeneChip Array (Affymetrix) was used for expression analysis during seed development. RNA from three (for dash mutant analysis) and four (for DASH ectopic expression analysis) independent biological replicates were analysed for each time point. Probe synthesis/labelling was carried out from 500 ng of RNA using the GeneChip 3?IVT express kit, according to the manufacturer?s instructions (Affymetrix). Array hybridization, scanning, and data normalization were performed as described by Benedito et al., (2008). Each file from the hybridized Affymetrix array was exported from GeneChip Operating Software version 1.4 (Affymetrix) and imported into Robust Multiarray Average Express (Irizarry et al., 2003) for global normalization. Presence/absence call for each probe set to remove background noise was obtained using dCHIP (Li and Wong, 2001). To identify probe sets differentially expressed in dash mutant vs WT control, the R package Anapuce (J. Aubert, UMR 518 AgroParisTech/INRA) was used. For each probe set, a paired t-test was performed on the log2 expression data from three arrays (3 independent biological repeats for 8 dap data), assuming that the variance of the log expression was the same for all transcripts per genotype. Spots with extreme specific variance, too low or too high, were excluded from the statistical analysis (details on the procedure given in Gagnot et al., 2008). P-values were adjusted by the Benjamini-Hochberg method (Benjamini and Hochberg, 1995), which controls the family-wise error rate.
Project description:To find downstream target of SOC1, we attemted global expression profiles in gain-of-function mutants of SOC1 To find downstream target of SOC1, we attemted global expression profiles in soc1-101D: Expt. 1 GSM73643, GSM73646 Expt. 2 GSM73647, GSM73648 Expt. 3 GSM73649, GSM73650 To find downstream target of SOC1, we attemted global expression profiles in soc1-2: GSM73649, GSM73651 To find downstream target of SOC1, we attemted global expression profiles in gain-of-function mutants of SOC1: GSM73643, GSM73646, GSM73647, GSM73648, GSM73649, GSM73650 Keywords: genetic modification
Project description:Comparison between transcriptomes of dash mutants vs WT plants and ectopic expression of 35S::DASH in hairy roots versus empty vector
Project description:Four-day old seedlings of Arabidopsis wildtype Col-0, T-DNA insertional mutant ataf2-2, and overexpression line ATAF2ox-3 were collected for total RNA extraction. Three biological replicates were prepared for each genotype.
Project description:The metabolome profiles of S. cerevisiae loss-of-function mutants in genes predicted to code for peroxisomally localized proteins were measured by flow-injection analysis.
Project description:We have shown that Notch signalling pathway is a potent regulator of thymic epithelial progenitor cell differentiation. Here we generated RNA-sequencing profile of early fetal TECs expressing low, normal or high levels of Notch. We found that loss of Notch function has no significant effects on the E12.5 TEC transcriptome. In contrast, loss and gain-of-function of Notch result in considerable changes in the E14.5 TEC transcriptome. This study uncovered a role of Notch in regulating cell state in TEC differentiation, as well as target genes and pathways regulated by Notch in this context.
Project description:The RNA-binding protein FUS/TLS, mutation in which is causative of the fatal motor neuron disease ALS, is demonstrated to directly bind to the U1-snRNP and SMN complexes. ALS-causative mutations in FUS/TLS are shown to abnormally enhance their interaction with SMN and reduce interaction with U1-snRNP. Correspondingly, global RNA analysis reveals a mutant-dependent loss of splicing activity, with ALS-linked mutants failing to reverse changes caused by loss of wild-type FUS/TLS. Furthermore, a common FUS/TLS mutant-associated RNA splicing signature is identified in ALS patient fibroblasts. Taken together, our studies establish potentially converging disease mechanisms in ALS and spinal muscular atrophy, with ALS-causative mutants acquiring properties representing both gain (dysregulation of SMN) and loss (reduced RNA processing mediated by U1-snRNP) of function. RNA-mediated oligonucleotide Annealing, Selection, and Ligation with Next-Generation sequencing (RASL-seq) method was used for analyzing alternative splicing changes. Oligonucleotide probes are designed to anneal to the exon-exon junctions. The probe library was assembled to assess 5530 unique alternative splicing events, most of which were exon inclusion or skipping, with a minority for alternative 5’- or 3’- splice sites. The splicing changes were compared among groups of reducing FUS/TLS or SMN levels, or expressing various FUS mutations to determine the loss versus gain of FUS/TLS function on splicing regulation.
Project description:Analysis of genes regulated by canonical Wnt signaling in the murine primary Schwann cells. Total RNA from b-catenin fl/fl Schwann cells, after introducing loss-of-function mutations with HTN-cre, or mimicking gain-of-function mutations with Chir98014 or Wnt3a-treatments, was compared to the respective controls.