ABSTRACT: Arabidopsis shoot apical meristem (SAM) stem cells were labelled with a CLV3:H2B-mCherry reporter. This reporter allowed fluorescence activated nuclear sorting (FANS) of nuclei of stem cells at 2 different developmental stages: 14d old seedling and 5 weeks old seedlings. DNA was extracted, bisulfite-converted and sequenced on an illumina HiSeq 2500 platform.
Project description:Arabidopsis shoot apical meristem (SAM) stem cells were labelled with a CLV3:H2B-mCherry reporter. This reporter allowed fluorescence activated nuclear sorting (FANS) of nuclei of stem cells at 4 different developmental stages: Embryo (heart-torpedo), 7d old seedling, 14d old seedling and 5 weeks old seedlings. RNA was extracted, converted to cDNA and sequenced on an illumina HiSeq 2500 platform.
Project description:Tumours induce de novo steroidogenesis in immune cells. We wanted to define the gene expression identity of intratumoural steroidogenic immune cells and compare them with the transcriptome with non-steroidogenic cells. Cyp11a1 expression is a faithful biomarker of de novo steroidogenesis (i.e. Cyp11a1+ cells are steroidogenic and Cyp11a1- cells are non-steroidogenic). We inoculated B16-F10 tumor in Cyp11a1-mCherry reporter mice, enriched and purified intratumoral Cyp11a1-mCherry+ and Cyp11a1-mCherry- cells by cell sorting into 96-well plates [with a ratio of 79:15 (mCherry+ : mCherry-) cells per plate] and performed scRNA-seq using SMART-Seq2 platform.
Project description:Beta cells are the sole source of insulin in our body, yet we do not understand how they mature into fully functional, glucose-responsive beta cells. We generated transcriptomes of FACS-purified beta cells using the Ins1-H2b-mCherry reporter line (Jax # 028589) at different peri- and postnatal maturation stages. This enables a systematic comparison across thousands of genes as beta cells mature.
Project description:Whole L1 larvae were collected from GC1459 [naSi2 [pGC550 (mex-5p::mCherry::H2B::nos-2 3'UTR +unc-119(+))] II; unc-119(ed3) III ?; daf-18(ok480) IV] and GC1171 [naSi2 [pGC550 (mex-5p::mCherry::H2B::nos-2 3'UTR +unc-119(+))] II; unc-119(ed3) III] strains up to two hours after hatching without food.
Project description:The goal of this experiment was to investigate the molecular mechanism of how Set-beta regulates neurite growth. Set-betaM-bM-^@M-^Ys subcellular localization is regulated by posttranslational modifications. We found that Set-beta suppresses neurite growth of purified postnatal rat retinal ganglion cell (RGC) primary neurons when it is overexpressed in the nucleus, whereas recruiting Set-beta to cellular membrane by fusing myr-tag to its N-terminus promotes neurite growth. Here, we transfected purified by immunopanning postnatal rat RGC with wild-type Set-beta which localizes to the nucleus, myr-Set-beta which is recruited to cellular membranes, and mCherry control, and analyzed with microarrays Set-betaM-bM-^@M-^Ys subcellular localization-dependent effects on gene expression. We found that wild-type Set-M-NM-2 regulated expression of significantly more genes than myr-Set-M-NM-2, consistent with wild-type Set-M-NM-2M-bM-^@M-^Ys nuclear localization and previously described roles in regulating transcription. These data reveal potential downstream gene effectors regulating neurite growth, and specific candidate genes could be validated and tested in future experiments. Acutely purified by immunopanning postnatal day 4 (P4) retinal ganglion cells (RGCs) transfected with mCherry, wild-type Set-M-NM-2, and myr-Set-M-NM-2, and co-transfected with pMAX-GFP (Lonza) constructs, were plated at high density on uncoated glass Lab-Tek II chamber slides (Thermo Fisher Scientific). The following morning, after gentle trituration, GFP-positive cells were FACS-isolated at 37M-BM-0C in PBS with BD FACSAria I cell sorter at 20 psi using a 130 M-BM-5m nozzle; GFP-negative threshold was set by first processing untransfected RGCs. Cells were gated on a FSC-A versus SSC-A plot to exclude dead cells and/or debris, and aggregates were removed using single cell gating with FSC-H versus FSC-W and SSC-H versus SSC-W plots. Immediately after isolation, GFP-positive cells were diluted in pre-equilibrated growth medium, centrifuged 15 minutes at 80 g, resuspended in growth medium in a 48-well tissue culture plastic plate as above, and cultured 2 more days in growth medium, as above. The Quick-RNA MicroPrep kit (R1050, Zymo Research) was used for RNA extraction 3 days after transfection, according to the manufacturer's instructions. Total RNA were used as input for the whole transcriptome amplification Ovation Pico WTA System V2 kit (3302-12, NuGEN), the cDNA product was quantified with Nanodrop 8000 Spectrophotometer (Thermo Scientific) and its quality was examined with a Bioanalyzer 2100 using the Nano 6000 kit (Agilent). 5 M-BM-5g of cDNA product from each sample was used as input for fragmentation and labeling using the Encore Biotin Module kit (4200-12, NuGEN); the fragmentation product quality was tested as above. The labeled product was hybridized to whole genome GeneChip Rat Gene 2.0 ST Arrays (Affymetrix). The staining, washing and scanning of the arrays was carried out using a Fluidics 450 station, GeneChip Operating Software and GeneChip Scanner 3000 7G (Affymetrix). Image intensities were analyzed with Expression Console (Affymetrix) from CEL files for quality control using standard GeneChip Rat Gene 2.0 ST Array control probes (Affymetrix), and to determine the RMA expression values and for annotations. RMA expression values were normalized to the median for each sample.
Project description:Purpose: To identify molecular pathways underlying epigenetic reprogramming in early germ cell precursors, we examined global gene expression of wild type primordial germ cells using mRNA sequencing. Methods: Given the limited number of PGCs collected from E9.5 to E13.5 (ranging from 300 to 5000), we used a low-input RNA sequencing method, Smart-Seq. RNA libraries were pooled and sequenced by Illumina Hiseq. Results: We generated >22 million uniquely mapped reads per sample and identified >10 thousand transcripts per genotype (RPKM>0.1). Hierarchical clustering and correlation analysis on gene expression indicates samples were clearly separated according to their genotypes with Spearman correlation coefficient of 0.98/0.99 within biological replicates. Compared with E9.5 PGCs, 479 genes were significantly up-regulated and 248 genes were down-regulated in E11.5 PGCs. When compared with E11.5 PGCs, male E13.5 PGCs had 362 up-regulated, and 239 down-regulated genes, whereas female E13.5 PGCs had 1163 up-regulated and 333 down-regulated genes. Overall, the number of up-regulated genes was greater than that of the down-regulated genes in every comparison, suggesting that gene expression is generally activated during PGC reprogramming. mRNA profiles of primordial germ cells derived from developmental embryos stages (E9.5, E11.5 and E13.5) were generated by deep sequencing, in duplicates (E9.5 and E11.5) or triplicates (E13.5f and E13.5m), using Illumina Hiseq.
Project description:Ongoing, lifelong neurogenesis maintains the neuronal population of the olfactory epithelium in the face of piecemeal neuronal turnover and restores it following wholesale loss. The molecular phenotypes corresponding to different stages along the progression from multipotent globose basal cell (GBC) progenitor to differentiated olfactory sensory neuron are poorly characterized. We used the transgenic expression of GFP and cell surface markers to FACS-isolate Sox2-GFP(+) GBCs, Neurog1-GFP(+) GBCs and immature neurons, and OMP-GFP(+) mature neurons from normal adult mice. In addition, the latter two populations were also collected 3 weeks after olfactory bulb ablation, a lesion that results in persistently elevated neurogenesis. Global profiling of mRNA from the populations indicates that all stages of neurogenesis share a cohort of >2100 genes that are upregulated compared to sustentacular cells. A further cohort of >1200 genes are specifically upregulated in GBCs as compared to sustentacular cells and differentiated neurons. The increased rate of neurogenesis caused by olfactory bulbectomy had little effect on the transcriptional profile of the Neurog1-GFP(+) population. In contrast, the abbreviated lifespan of OMP-GFP(+) neurons born in the absence of the bulb correlated with substantial differences in gene expression as compared to the mature neurons of the normal epithelium. Detailed examination of the specific genes upregulated in the different progenitor populations revealed that the chromatin modifying complex proteins LSD1 and coREST were expressed sequentially in upstream Sox2-GFP(+) GBCs and Neurog1-GFP(+) GBCs/immature neurons. The expression patterns of these proteins are dynamically regulated after activation of the epithelium by methyl bromide lesion. Total RNA was isolated from dissected, dissociated and FACS-purified olfactory mucosal cells from normal adult mice or mice 3 weeks after unilateral bulbectomy. Cells were purified with FACS using endogenous GFP fluorescence from various transgenic lines and cell surface labeling. Two to seven adult mice were used per replicate and three replicates per condition were performed using Illumina bead arrays. 21 samples from olfactory mucosa were analyzed in this series and 3 samples were from a commercially available reference RNA sample. Universal mouse reference RNA from Stratagene was used as a general control and Normal olfactory mucosa was used as a tissue specific control.
Project description:We performed an experimental Cas13d-SARScov2 genome-wide screen to identify gRNAs that would allow Cas13d to degrade the viral RNA. We built mCherry reporter plasmids that express mCherry with a 3kb 3'UTR deriving from the SARScov2 genome. In total we designed 11 reporters covering the entire plus strand of the viral genome and 11 other reporters covering the entire minus strand. Each of the 22 mCherry reporter plasmids carries a U6 expression cassette containing a Cas13d gRNA that targets the 3'UTR of the mCherry reporter. Each reporter is represented by a pool of reporters each containing a different gRNA that targets mCherry 3'UTR for a total average of ~300 gRNA per 3'UTR. The entire pool of 22 reporters, each with a pool of ~300 different gRNAs constitutes a comprehensive set ~6,500 reporters (~ 6,500 different gRNAs) that allowed us to interrogate the entire SARScov2 plus and minus strand viral RNA for regions of vulnerability and targetability. In order to specifically interrogate Cas13d activity an remove the biases that would be introduced in the reporter expression by the presence of a large 3kb 3'UTR we used a case (presence of Cas13d) control (absence of Cas13d) design. Briefly, the ~6,500 reporters were lentiviral transduced in RKO cells, the cells were split in 2 populations, 1 population was transduced with Cas13d and the other serving as control did not. The population expressing Cas13d was FACS sorted in low mCherry (efficient gRNAs) and high mCherry (un-efficient gRNAs) in 2 biological replicates and the genomic DNA of these populations was extracted, gRNAs were PCR amplified and sequenced. For the population that did not express Cas13d, a low mCherry, one high mCherry and unsorted population were sequenced as control libraries.
Project description:NvElav1::mOrange is a stable transgenic line that labels a large fraction of the nervous system of the sea anemone Nematostella vectensis (Nakanishi et al., Development 2012). Two week old primary polyps were dissociated and the NvElav1::mOrange positive cells were enriched by FACS.
Project description:The goal of the study is to analyze the function of 3'-UTR motifs in the non-native context. Several 3'-UTR motifs predicted to have stabilizing or destabilizing effects on mRNA were inserted into terminators controlling the expression of reporter mCherry gene in following contexts: pRps3-mCherry-tRps3, pPgk1-mCherry-tRps3, pTsa1-mCherry-tTsa. 3'-end sequencing was used to measure the levels of expression of reporter gene and distribution of poly(A) sites.