Next Generation Sequencing to Study FLK-affected genes
Ontology highlight
ABSTRACT: Purpose: The goals of this study are to compare RNA-seq profiles of Col-0, acd6-1, flk-1, flk-5, acd6-1flk-1, and acd6-1flk-5 to identify genes affected by FLK. Methods: Total RNA was extracted from 25-d old plants. A total amount of 1 μg RNA per sample was used to generate cDNA libraries, using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. Deep sequencing was performed using Illumina NovaSeq 6000 (Novogene Corporation Inc.). Triplicate biological samples were used for all genotypes except acd6-1flk-5, which had duplicate samples because one sample failed to pass quality control. The samples were multiplexed and sequenced with the standard paired-end sequencing that has a read length of 150bp per end and 20M reads per end per sample. Results: We found that over 8000 genes are differentially affected by FLK based on the RNAseq analysis. GO analysis revealed that FLK-affected genes are enchriched for genes involved in primary metabolism and in response to abiotic and biotic stimuli. Conclusions: The RNAseq analysis support a role of FLK in regulation of plant development and defense.
Project description:Screening for genes up in Etv2+ cells within Flk-1+ ES derived mesoderm Microarray analysis performed to screen for the candidate genes regulated by Etv2. Differentiated Flk-1+ mesoderm can be devided into Etv2+ or-. Etv2+ cells are assumed to be committed to hemato/endothelial cells. Comparison of two populations can reveal genes relevant in this commitment.
Project description:Screening for genes up in Etv2+ cells within Flk-1+ ES derived mesoderm Microarray analysis performed to screen for the candidate genes regulated by Etv2. Differentiated Flk-1+ mesoderm can be devided into Etv2+ or-. Etv2+ cells are assumed to be committed to hemato/endothelial cells. Comparison of two populations can reveal genes relevant in this commitment. Extract RNA from sorted Flk-1+/Etv2- vs Flk-1+/Etv2+ populations.Etv2-Venus KI ES cells were differentiated on OP9 for 4-5 days and Flk-1+ population was separated into Etv2-Venus+ or- cells. Total RNA was purified from each population for analysis.
Project description:Screening for genes regulated by Etv2 within Flk-1+/PDGFRa+ ES derived mesoderm.Microarray analysis performed to screen for the candidate genes regulated by Etv2. TT2 ES cells differentiated on OP9 feeder cells were sorted using Flk-1 and PDGFRa antibodies.Gene expressions from these two populations were compared.
Project description:Screening for genes regulated by Etv2 within Flk-1+/PDGFRa+ ES derived mesoderm.Microarray analysis performed to screen for the candidate genes regulated by Etv2. TT2 ES cells differentiated on OP9 feeder cells were sorted using Flk-1 and PDGFRa antibodies.Gene expressions from these two populations were compared. Extract RNA from sorted Flk-1+/PDGFRa+ populations from Etv2Het vs KO cells. To obtain primitive mesoderm cells TT2 ES cells of corresponding genotypes were differentiated on OP9 cells for 4 days. Flk-1+/PDGFRa+ populations were sorted from Etv2 Het vs. KO cells for RNA extraction.
Project description:Purpose: RNA-Seq uses next-generation sequencing to analyze expression across the transcriptome, enabling to detect known or novel features and quantify RNA. The goal of this study is to compare transcriptome profiles between control and GLUT1-HK2-PFKFB3 overexpressed mouse skeletal muscle. Methods: Total RNA was isolated from the gastrocnemius muscle of chow diet-fed M;G and control mice at the age of 3-month-old using RNAiso Plus (Takara Bio). Three independent pooled samples per group were analyzed. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and 125 bp/150 bp paired-end reads were generated. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. Fragments Per Kb of exon per Million mapped reads (FPKM) were calculated using featureCounts v1.5.0-p3. Results: We mapped about 6 million sequence reads per sample to the mouse genome and identified 54533 transcripts in the gastrocnemius muscle of control and GLUT1-HK2-PFKFB3-overexpressed mice. RNA-seq data confirmed stable expression of 10 known housekeeping genes, and 7 of these were validated with qRT–PCR. A total 664 transtripts were differentially expressed in wildtype and GLUT1-HK2-PFKFB3 overexpressed skeletal muscle with a cutoff of 2.0-fold and p < 0.05 from RNA sequencing analysis. Altered expression of 15 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method.
Project description:ES derived Flk-1+ cells were separated by sorting into Hoxb6 positive and negative populations by Venus reporter. Gene expression was compared between these two groups.
Project description:ES derived Flk-1+ cells were separated by sorting into Hoxb6 positive and negative populations by Venus reporter. Gene expression was compared between these two groups. Duplicate analysis of Hoxb6-Venus positive and negative Flk-1+ cells.
Project description:ES derived Flk-1+ cells were separated by sorting into Hoxb6 positive and negative populations by RFP Hoxb6 reporter. Gene expression was compared between these two groups.
Project description:ES derived Flk-1+ cells were separated by sorting into Hoxb6 positive and negative populations by RFP Hoxb6 reporter. Gene expression was compared between these two groups. Duplicate analysis of Hoxb6-RFP positive and negative Flk-1+ cells.