Project description:Understanding the complex effects of genetic perturbations on cellular state and fitness in human pluripotent stem cells (hPSCs) has been challenging using traditional pooled screening techniques which typically rely on unidimensional phenotypic readouts. Here, we use barcoded open reading frame (ORF) overexpression libraries with a coupled single-cell RNA sequencing (scRNA-seq) and fitness screening approach, a technique we call SEUSS (ScalablE fUnctional Screening by Sequencing), to establish a comprehensive assaying platform. Using this system, we perturbed hPSCs with a library of developmentally critical transcription factors (TFs), and assayed the impact of TF overexpression on fitness and transcriptomic cell state across multiple media conditions. We further leveraged the versatility of the ORF library approach to systematically assay mutant gene libraries and also whole gene families. From the transcriptomic responses, we built genetic co-perturbation networks to identify key altered gene modules. Strikingly, we found that KLF4 and SNAI2 have opposing effects on the pluripotency gene module, highlighting the power of our method to characterize the effects of genetic perturbations. From the fitness responses, we identified ETV2 as a driver of reprogramming towards an endothelial-like state.
2018-09-24 | GSE107185 | GEO
Project description:oRiboT-Fitness-Landscape
| PRJNA927307 | ENA
Project description:A Genetically Encoded Device for Transcriptome Storage
Project description:International guidelines recommend deciding the treatment of colorectal lesions based on the estimated histology by endoscopic optical diagnosis. However, the theoretical and practical knowledge on optical diagnosis is not widely expanded
The mail goal of this randomised controlled trial is to compare the pooled sensitivity of optical diagnosis for predicting deep submucosal invasion in large non-pedunculated polyps > 20 mm assessed in routine colonoscopies of gastroenterologists attending a e-learning module (intervention group) vs gastroenterologists who do not (control group)
The main questions the study aims to answer are:
* Is the pooled sensitivity of optical diagnosis for predicting deep submucosal invasion in large non-pedunculated polyps assessed in routine colonoscopies increased in those gastroenterologists participating in the e-learning module?
* Is the pooled diagnostic accuracy of optical diagnosis for predicting deep sm invasion in large non-pedunculated polyps ≥ 20 mm assessed in routine colonoscopies increased in those gastroenterologists participating in the e-learning module?
* In lesions with submucosal invasion, is the en bloc and complete resection rate (R0) increased in those gastroenterologists participating in the e-learning module?
* In lesions referred to surgery, is the pooled benign polyps rate decreased in those gastroenterologists participating in the e-learning module?
* In lesions treated with advanced en bloc procedures (ESD, TAMIS, fullthickness resection), is the pooled rate of histology with high-grade dysplasia, intramucosal cancer or submucosal invasion increased in those gastroenterologists participating in the e-learning module?
* In lesions treated with piecemeal endoscopic resection, is the pooled rate of histology with high-grade dysplasia, intramucosal cancer or submucosal invasion decreased in those gastroenterologists participating in the e-learning module?
* Is the diagnostic accuracy for predicting deep submucosal invasion in a test with pictures increased after participating in the e-learning module?
The participants (or subjects of study) are gastroenterologists. They will be randomised to do the e-learning course (intervention group) or not (control group).
Researchers will compare clinical outcomes of gastroenterologists participating in the e-learning module vs gastroenterologists not participating in the e-learning module to see if:
* the pooled sensitivity of optical diagnosis for predicting deep submucosal invasion in large non-pedunculated polyps > 20 mm assessed in routine colonoscopies is increased.
* the pooled diagnostic accuracy of optical diagnosis for predicting deep sm invasion in large non-pedunculated polyps > 20 mm is increased.
* the en bloc and complete resection rate (R0) is increased in lesions with submucosal invasion.
* the pooled benign polyps rate decreased in lesions referred to surgery.
* the pooled rate of histology with high-grade dysplasia, intramucosal cancer or submucosal invasion increased in lesions treated with advanced en bloc procedures (ESD, TAMIS, fullthickness resection).
* the pooled rate of histology with high-grade dysplasia, intramucosal cancer or submucosal invasion decreased in lesions treated with piecemeal endoscopic resection.
* the diagnostic accuracy for predicting deep submucosal invasion in a test with pictures after participating is increased.
Project description:To identify genes that are downstream of gonadal hormones and that control dimorphic behaviors, we used a MEEBO array platform to profile gene expression of adult male and female hypothalamus against a whole brain reference sample. The experimental design allowed us to identify genes that are upreguated in the hypothalamus compared to the whole brain and are dimorphically expressed between the two sexes. In situ hybridization of candiate genes were carried out to validate the dimorphic expression of these genes in the hypothalamus. Array results were used to create a list of genes to screen by in situ hybridization. For each normalization method, a list of genes that were upregulated in the male or the female was created, and a gene had to be on a set number of these individual normalization lists in order to be considered for screening. A similar method was used to create a list of hypothalamic upregulated genes. The final screening list was comprised of genes that were upregulated in the male or female as well as in the hypothalamus. If in situ hybridization proved that the gene was upregulated in the male or female brain, we concluded that that transcript was differentially expressed. No one normalization method was weighted over another, and the array results were used to create a screening list for in situ hybridizations. Hypothalami from 4 adult animals of each sex were microdissected and pooled for each experimental sample and the whole brain plus pituitary from one male and one female were pooled to provide the reference sample. Each set of samples (male and female) was hybridized to two arrays to provide two technical replicates for each set of samples taken. A total of three sets of samples were taken (three biological replicates). For the "_amp" Samples, source (hypothalamus and whole brain) mRNA was amplified using T7 based method. For this set, data were not normalized, rather the raw intensities were used to calculate ratios. Candidate genes from this study was also included in final list in situ screening.
Project description:"Position effect" means that the activity of an integrated reporter gene (IR) depends on its genomic location. Although the impact of local environment on IR (cis-effect of location) has been widely reported, the impact of IR integration on local environment and its potential mechanism (reaction of location), such as the change of expression landscape and its fitness are poorly understood. Here, we randomly selected ~250 previously constructed strains with GFP gene inserted into hundreds of different locations separately in yeast, and performed high-throughput transcriptome sequencing.