AgRP1::ChR2-Kaede
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ABSTRACT:
PROVIDER: PRJEB85762 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:We performed Illumina sequencing of sRNA libraries prepared from leaves of adult plants in Arabidopsis. Chromatin remodeling factor 2 (CHR2) positively regulates transcription of MIR loci whereas repressing microRNA (miRNA) accumulation in vivo. CHR2 can directly bind to and unwind primary miRNAs (pri-miRNAs) and inhibit their processing; and this inhibition entails its remodeling activity in vitro and in vivo.
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Project description:We have developed a method for rapidly identifying early, regionally-expressed molecules: FACS-Assisted Microdissection of Photolabeled cells (FAM-P). For our first FAM-P project, we injected embryos with purified Kaede protein, a stony coral protein that fluoresces at 514 nm (green) prior to- and 582 nm (red) after photoconversion at 405 nm. We used the scanning laser of a confocal microscope to photolabel late blastula-stage embryos along 4-6 tiers of marginal cells, comprising the mesoderm and endoderm germ layer precursors. This procedure left the neurectoderm precursor cells marked by green fluorescence and the mesendoderm precursor cells marked by red fluorescence. Embryonic cells were dissociated and subjected to FACS, separating red mesendoderm precursors from green neurectoderm precursors. RNA was extracted and linearly amplified once, then dye-labeled and co-hybridized to a microarray with over 30,000 oligos representing more than 20,000 unique zebrafish genes. In validation of our strategy, many genes known to have elevated expression in the late blastula margin (e.g., squint, fgf8, lim1 and gata5) and several genes known to be specific to the early neurectoderm (e.g., sox31 and otx2) were independently identified by this approach. In addition to known genes, our method has identified dozens of previously uncharacterized genes that are enriched in the pre-mesendoderm or pre-neurectoderm. Keywords: 40% epiboly-stage embryos, injected with purified Kaede protein, differentially photolabeled, disaggregated and FACS sorted 34k expression arrays were generated by printing Oligo nucleotides from three different sets (MWG, Compugen and Operon) onto epoxy slides. Total RNA from Kaede protein-injected and FAM-P treated zebrafish embryos was amplified using an Ambion kit (Cat#1753), labeled with Cy dyes and hybridized overnight to the oligo chip in a MAUI chamber. The experiment comparing mesendoderm was performed four times (twice with WIK/AB hybrid strain embryos, once with EK strain embryos and once with AB strain embryos) each time including a dye swap, for a total of eight (four forward and four dye-swap) hybridizations. Three control cRNA probes were also generated from AB-strain embryos, namely: (1) whole 40% epiboly stage embryos, (2) FACS-sorted green cells from dissociated 40% epiboly embryos that were Kaede-injected but NOT photolabeled and (3) FACS-sorted red cells from dissociated 40% epiboly embryos that were Kaede-injected AND photolabeled. These probes were each generated three times, each time including a dye swap and co-hybridized as follows: (1) whole embryo cRNA vs green cell cRNA (three forward and three dye-swap hybridiizations) and (2) green cell cRNA vs. red cell cRNA (three forward and three dye-swap hybridizations).
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Project description:Here we present dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) prepared from leaves of adult plants in Arabidopsis. Chromatin remodeling factor 2 (CHR2) positively regulates transcription of MIR loci whereas repressing microRNA (miRNA) accumulation in vivo. CHR2 can directly bind to and unwind primary miRNAs (pri-miRNAs) and inhibit their processing; and this inhibition entails its remodeling activity in vitro and in vivo.
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Project description:Preterm children are frequently exposed to various painful stimuli in the early days of life. Early-life-pain (ELP) may affect cortical development and reduce the fitness and resilience in during youth and adult life. We developed an optogenetic Cre/loxP mouse model of "early-life-pain" (ELP) using mice with transgenic expression of an improved channelrhodopsin-2 (ChR2)/Td-Tomato fusion protein under control of the advillin promoter, that drives expression of ChR2/td-Tomato specifically in primary somatosensory neurons of the dorsal root ganglia (Avil-ChR2 mice). Floxed ChR2/td-Tomato littermates (ChR2-flfl) were used as controls. Mice were exposed to blue light in a chamber once daily from postnatal day P1-P5. Cortical gene expression was analyzed at P7 via RNA-sequencing in eight biological replicates per group. Cortices were rapidly excised and snap froze in liquid nitrogen. Total RNA was extracted from with Qiagen RNeays spin columns and quantified on a Nanodrop. Sequencing libraries were prepared using Illumina TruSeq stranded mRNA library preparation kit, and sequencing was performed on an Illumina NGS platform. The alignment was performed with SeqMan NGen 17 (Lasergene) using the reference genome mm10 provided from UCSC (GRCm38) as template, a minimum read length of 35 bp and automatic adapter trimming. Sequence reads were normalized with EdgeR and analyzed in ArrayStar 17 (Lasergene). Differential gene expression was assessed with general linear models and adjusted according to Benjamini Hochberg.
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Project description:We wanted to query the differences and similarities between our wild-type C2C12 cell line and our optogenetic ChR2 C2C12 myoblasts on the same day of differentiation. After 6 days on muscle differentiation medium, we extracted the RNA and sequenced it for 6 wells each.
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